bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Aging on Angiogenic T cells
Authors: Kushner Erich J , MacEneaney Owen J , Weil Brian R , Morgan Richard G , Mestek Micheal L , Van Guilder Gary P , Greiner Jared J , Diehl Kyle J , Stauffer Brian L , DeSouza Christopher A
Published: June 2021

CD31+ T cells, or angiogenic T cells, exhibit vasculoprotective and neovasculogenic qualities. Aging is associated with reduced angiogenic capacity and increased cardiovascular risk. Currently, the influence of age on CD31+ T cell number and function is unclear. We tested the hypothesis that circulating CD31+ T cell number and migratory capacity are reduced, while apoptotic susceptibility is heightened, with advancing age in adult humans. CD31+ T cells were isolated by magnet‐activated cell sorting from 31 healthy, sedentary men: 10 young (Y: age 25±1 yr), 12 middle‐aged (MA: age 47±1 yr) and 9 older (O: 64±2 yr). Fluorescence‐activated cells sorting analysis revealed that the number of circulating CD31+ T cells was lower (p<0.01) in O (24% of CD3+ cells) compared with MA (38% of CD3+ cells) and Y (40% of CD3+ cells) men; however, there was no difference between MA and Y. The ability of CD31+ T cells to migrate (boyden chamber) to both VEGF and SDF‐1α was significantly reduced in MA (310±51 and 292±43 AU) and O (255±100 and 300±138 AU) compared with Y (540±86 and 601±97 AU) men. No group differences were detected in intracellular active caspase‐3 or cytochrome‐c levels in response to staurosporine stimulation. These results indicate that aging is associated with both diminished CD31+ T cell number and migratory function in healthy men.
Gastrocnemius fatigue is increased and blood flow is reduced in adult skeletal myocyte‐specific VEGF‐deficient mice
Authors: Goldberg Daniel B , Tang Kechun , Wagner Peter D. , Breen Ellen C.
Published: June 2021

Adult skeletal muscle‐specific VEGF gene‐deleted mice demonstrate exercise limitation, but muscle capillarity is maintained. To determine if exercise impairment is due to reduced skeletal muscle blood flow, gastrocnemius flow at rest and during electrically stimulated (ES) muscle contraction was assessed with fluorescent microspheres. Myocyte‐specific VEGF gene deletion was tamoxifen‐induced (D0‐D4, i.p.) in 4 month old VEGFLoxP X HSA‐Cre‐ERT2(+) and (−) mice. On day 21 post‐tamoxifen gastrocnemius VEGF is reduced by 94%. At this time point the gastrocnemius complex of one leg was stimulated via the sciatic nerve (10 volts) and force monitored by a pressure transducer connected to the tendon. 15 μ microspheres (0.2 ml, 1 x 106 beads/ml) were delivered into the aorta via the left carotid artery during the first minute of contractions. VEGFLoxP X HSA‐Cre‐ERT2(+) mice had 76% less resting gastrocnemius flow than control (Cre −). Only control mice increased blood flow during ES (by 60%). Time to reach 50% of initial force was reduced in VEGF‐deficient mice [156+/−31 (sd) sec.] compared to controls [475+/−246 (sd) sec.]. These data suggest that reduced skeletal myocyte‐specific VEGF expression in adult mice attenuates both basal and contraction‐induced blood flow, and this vascular alteration may contribute to the greater muscle fatigue.
Impaired vascular reactivity following chronic ischemia in arteries of the mouse hindlimb
Authors: Cardinal Trevor R. , Kesler Thomas J.
Published: June 2021

Collateral‐dependent hyperemia is reduced following chronic ischemia, but the contribution of individual vessel reactivity is unknown. Further, it is not known what aspect of the complex ischemic injury response impacts vascular reactivity. To determine the impact of ischemia on vascular reactivity, we measured functional vasodilation in the muscular branch artery following resection of the femoral artery proximal to the muscular branch, which induces ischemia in the muscular branch artery. On day‐14 after surgery the diameter of the muscular branch was measured using side‐stream dark field (SDF) imaging intravital microscopy. At moderate intensity skeletal muscle contraction (1mA, 200μs, 8Hz, 90sec), functional vasodilation is reduced compared to the contralateral limb (10.84 ± 8.79% versus 151.70 ± 22.15% increase above baseline). Conversely, at high intensity skeletal muscle contraction (1mA, 500μs, 8Hz, 90sec), the initial vasodilation is not different from the contralateral limb, but the ischemic arteries are refractory to regaining their resting diameter. The control artery returned to baseline diameter within 10‐minutes following high‐intensity muscle contraction, while the ischemic artery did not return to baseline after 25 minutes. Further investigation is underway to determine if endothelial or smooth muscle dysfunction underlies this abnormal reactivity in ischemic arteries.
Restoration of angiogenesis phenotype in new congenic lines based on Dahl salt‐sensitive (SS/Mcwi) background
Authors: Stodola Timothy , de Resende Micheline , Moreno‐Quinn Carol , Greene Andrew
Published: June 2021

Angiotensin II is an important mediator of skeletal muscle angiogenesis induced by electrical stimulation. Previous studies have shown that substitution of BN chromosome 13 entirely and introgression of a 4.5 Mbp piece of BN chromosome 13 that overlaps with the renin gene into the Dahl salt‐sensitive (SS/Mcwi) rat restores renin levels and the angiogenic response to electrical stimulation (estim). In this study we explored the effects of substituting smaller pieces of the BN chromosome 13 located near the renin gene and tested them for restoration of the angiogenesis phenotype. Three new congenic lines were produced (A: 2.0 Mbp, overlaps renin, B: 0.9 Mbp, no overlap with renin upstream, C: 2.6 Mbp, no overlap with renin downstream). Electrical stimulators were implanted to intermittently stimulate the peroneal nerve in one hind limb, contracting the Tibialis Anterior (TA) and Extensor Digitorum Longus (EDL) muscles for 8 hours/day for 7 days. Line B had no increase in vessel density, while both Line A and C had an increase in vessel density due to estim. These data support the concept of trans regulation of renin by elements captured in line 9c. Supported by NIH Heart Lung & Blood Institute 082798.
Neurovascular alignment in adult rat mesenteric microvascular networks
Authors: Stapor Peter C , Murfee Walter Lee
Published: June 2021

An emerging area of microvascular research focuses on the link between neural and vascular patterning. The link is supported by overlapping molecular signals, however, the spatial and temporal coordination between the two systems at the microcirculation level in the adult remains under‐investigated. The objective of this study was to quantify the neurovascular alignment across the hierarchy of adult microvascular networks containing both systems. Mesentery tissues were harvested from 340–440 g male Wistar rats and immunolabeled for PECAM, an endothelial cell marker, and NG2, a vascular pericyte marker, which also identifies nerves. From four microvascular networks, neurovascular alignment was quantified per five vessel categories: arterioles (>20 μm), pre‐capillary arterioles (10–20 μm), capillaries (<10 μm), post‐capillary venules (10–20 μm), and venules (>20 μm). Blood vessels and nerves were considered aligned when they were within 10 μm of each other for at least 50 μm. 52% of the total vascular length aligned with nerves. 97% of the arteriole length, 74% of pre‐capillary arteriole length, 38% of the capillary length, 57% of the post‐capillary venule length, and 76% of the venule length aligned with nerves. The observations suggest that neurovascular alignment exists in adult rat microvascular networks, and establish a model to investigate the functional coordination between both systems.
Mouse models of variability in vascular remodeling: collateral networks in spinotrapezius muscle ischemia
Authors: Mac Gabhann Feilim , Peirce Shayn M.
Published: June 2021

We used inbred and outbred mouse strains as models for interindividual variability in microvascular network structure. Other groups previously identified remodeling responses of mouse strains to ischemic ligation of hindlimb arteries. Our studies induced arterial ischemia in the mouse spinotrapezius, a model system that allows for the whole‐mount, en‐face visualization of the entire muscle microvasculature. We observed two groups of mice: ischemia‐vulnerable, characterized by dendritic arteriolar structure, and ischemia‐protected, characterized by frequent arteriole‐to‐arteriole connections. During vascular remodeling, mice that were characterized as “ischemia‐vulnerable” re‐routed blood to the ischemic tissue via the capillaries, a subset of which increased in diameter and became arterialized. Mice in the “ischemia‐protected” population re‐routed blood flow via arterioles, which showed tortuosity and diameter increases, while capillaries were unchanged. Each outbred CD1 mouse fell into one of the above two categories, siblings having different vascular phenotypes. We developed a computational model to quantify the protective effect of arteriole‐to‐arteriole connections, and found that only a small number of these higher‐diameter bridges are needed to provide protection to the tissue. Support provided by: NIH‐HL082838‐02, NIH‐T32‐HL007284 and NIH‐K99‐HL093219.
The effects of bosentan on thrombin‐activated fibroblasts and endothelial cells
Authors: Bogatkevich Galina S , Ludwicka‐Bradley Anna , Silver Richard M
Published: June 2021

Endothelin‐1 and thrombin are elevated during lung injury and repair, as seen in scleroderma and other interstitial lung diseases, and each plays important roles in remodeling epithelium, blood vessels and connective tissue. Both factors promote myofibroblast differentiation, the hallmark of pulmonary fibrosis. Here we show that thrombin significantly induces endothelin‐1 expression in human lung fibroblasts. Bosentan, the dual, specific and competitive inhibitor of endothelin, significantly decreases α‐SMA, collagen gel contraction, and proliferation of human lung fibroblasts stimulated by thrombin, suggesting that thrombin‐induced differentiation of fibroblasts to the myofibroblast phenotype is at least in part regulated by endothelin‐1. Moreover, bosentan inhibits the basal level of fibroblast proliferation suggesting that endogenous endothelin may be in part responsible for fibroblast proliferation. The effect of bosentan on endothelial cells is concentration dependent. At concentrations of 10μM and 100μM bosentan inhibits thrombin‐induced endothelial cell migration and angiogenesis, whereas low‐dose (10nM) bosentan demonstrates a significant angiogenic effect. Bosentan rapidly phosphorylates Akt in endothelial cells and promotes cell survival. Our results demonstrate that low‐dose bosentan protects endothelial cells from apoptosis and increases their functionality. We conclude that bosentan may be a promising therapeutic agent in the treatment of microvascular injury as seen in scleroderma and other interstitial lung diseases.

Study sponsor: Actelion Pharmaceuticals
Biological activity of VEGF in plasma from patients with pre‐eclampsia
Authors: Bates David O , Bills Victoria , Soothill Peter , Harper Steven J
Published: June 2021

In pre‐eclampsia VEGF‐A, a potent endothelial activator is upregulated as measured by radio and competitive‐enzyme immunoassays, and PlGF is reduced. However, the biological activity of VEGF in pre‐eclamptic plasma is not clear. To determine if circulating VEGF levels in pre‐eclampsia are biologically active, plasma from pre‐eclamptic (n=17) and normotensive (n=10) pregnant women was perfused into Rana mesenteric microvessels, and microvascular permeability measured using the Landis micro‐occlusion technique. Pre‐eclamptic but not normotensive plasma induced a mean±SEM 5.25±0.8 fold acute increase in vascular permeability (p=0.0003). This rise was blocked by bevacizumab, an antibody to VEGF (n=7, p=0.012), and by VEGF receptor inhibition by SU5416 at doses specific to VEGF receptor‐1, but not by the VEGF receptor‐2 inhibitor, ZM323881. There was no difference in VEGF levels in the pre‐eclamptic compared with the normal plasma measured by ELISA, but PlGF levels were significantly reduced. These findings suggest that circulating VEGF levels in pre‐eclampsia are biologically active and a lack of PlGF may be involved in the increased vascular permeability of pre‐eclampsia.
cAMP via Epac/Rap1 enhanced microvascular endothelial barrier functions by Rac1‐mediated reorganization of adherens and tight junctions
Authors: Peter Dominik , Spindler Volker , Waschke Jens
Published: June 2021

cAMP stabilizes the endothelial barrier, in part by activation of Rac1. However, the effects on cell junctions on the ultrastructural level are largely unknown. Here, in human dermal microvascular endothelial cells, we used forskolin/rolipram (F/R) to increase cAMP as well as O‐Me‐cAMP to stimulate Epac/Rap 1 signaling. Under both conditions, endothelial barrier properties were enhanced as detected by increased transendothelial electrical resistance (TER) which was paralleled by activation of Rac 1. Compared to controls, electron microscopy revealed a significantly higher frequency of complex intercellular junctions containing both adherens and tight junctions which may reflect the increase in VE‐cadherin and claudin 5 immunostaining intensity at cell borders. In contrast, cleft length and intercellular overlap were not significantly reduced on the ultrastructural level but adherens and tight junction immunostaining appeared to be lineralized indicating recruitment of adhesion molecules to and formation of junction strands within the intercellular cleft. Barrier stabilization and junction reorganization in response to O‐Me‐cAMP were completely abolished in the presence of NSC‐23766 which specifically interferes with Rac1 activation. These results indicate that Rac1‐mediated junction reorganization was required for Epac/Rap1‐mediated cAMP‐induced barrier stabilization. DFG SFB688, A4
Manipulation of smooth muscle BKCa using subunit directed siRNA
Authors: Yang Yan , Ella Srikanth R , Braun Andrew P , Korthuis Ronald J , Davis Michael J , Hill Michael A
Published: June 2021

Cremaster and cerebral vascular smooth muscle cells (VSMC) exhibit heterogeneity in large conductance Ca2+‐activated K+ channels (BKCa) partly due to differences in β1:α subunit ratio. To gain insight into BKCa methods were developed for subunit‐specific knockdown of the channel. Using transient transfection approaches and small interfering RNAs (siRNA) either the α or β1 subunit was targeted in isolated arterioles. Control studies used fluorescently labeled siRNA or unrelated siRNA. After 2–3 days culture fluorescence images and whole cell K+ currents were examined in dispersed VSMC. From functional data α‐subunit expression was reduced by ~60% in both vessels. Thus, at +70 mV, IBTX‐sensitive K+ current density was significantly reduced after α‐subunit siRNA compared to control. Similarly, STOC frequency (at +20 mV) decreased following siRNA treatment while BKCa opening by NS1619 or estrogen (E2) was decreased. Cells treated with β1‐subunit siRNA showed impaired responses to E2 with a greater effect in cerebral VSMCs compared to those of cremaster. Thus transient transfection and siRNA can be used to effectively decrease endogenous BKCa activity in intact small arteries. Further, cerebral VSMCs treated with β1‐subunit siRNA exhibit a functional phenotype similar to untreated cremaster VSMCs, supporting the idea that differences in β1:α subunit ratio underlie observed heterogeneity in BKCa activity.
EPACs Inhibit ATP Release from Human Erythrocytes (RBCs) in Response to Activation of GPCRs possibly via Activation of PKC.
Authors: Adderley Shaquria P , Sridharan Meera , Bowles Elizabeth , Stephenson Alan , Ellsworth Mary , Sprague Randy
Published: June 2021

Increases in cAMP are required for ATP release from RBCs and are regulated by phosphodiesterases (PDEs). Selective PDE inhibitors potentiate increases in cAMP and ATP in response to activation of either the prostacyclin or β adrenergic receptor in these cells. Here we demonstrate that non‐selective PDE inhibition attenuates ATP release in response to activation of either receptor. We hypothesize that increases in cAMP not associated with receptor activation stimulate exchange proteins activated by cAMP (EPACs) that inhibit ATP release via PKC activation. 8pCPT2OMecAMP (8pCPT, 10μM), a direct EPAC activator inhibited both iloprost (ILO 10nM, n=6)‐and isoproterenol (1μM, n=6)‐induced ATP release. To demonstrate that this inhibition was not due to activation of PKA, RBCs were incubated with 6BnzcAMP (30μM), a direct PKA activator, followed by ILO. PKA activation potentiated ATP release (n=2). To determine if 8pCPT‐induced inhibition of ATP release was via activation of PKC, RBCs were treated with PMA (1μM) prior to ILO. PMA inhibited ILO‐induced ATP release (n=6). 8pCPT also inhibited ATP release with forskolin (10μM, n=6), a direct activator of adenylyl cyclase, indicating that inhibition of ATP release is downstream of cAMP synthesis. These results support the hypothesis that increases in cAMP within RBCs can activate EPACs which inhibit ATP release, possibly via PKC activation. [NIH grants HL‐64180 & ‐89094]
Biocompatibility of crosslinked aerogels of variable densities with blood platelets and vascular endothelial cells
Authors: Morton Blake Edward , Jarrett Ellen , Lu Hongbing , Yin Wei , Rubenstein David Alan
Published: June 2021

Evaluating the vascular biocompatibility of crosslinked aerogels (X‐aerogels) can result in the development and improvement of blood implantable devices. X‐aerogels of variable densities can be used to optimize the overall compatibility of the material with respect to material processing. Initial research has shown that X‐aerogels do not alter platelet activation or aggregation. Here, platelets were incubated with X‐aerogel samples of different densities for up to 24 hours. Optical light aggregometry was used to quantify the rate and total extent of platelet aggregation at 0, 0.5, 1, 2, 4, 6, and 24 hours. Platelet activation state (PAS) was quantified by the modified prothrombinase assay under static and flow conditions, which measures thrombin generation. Flow cytometry was performed to quantify the surface protein expressions of CD41 and CD62P of platelets to identify a possible mechanism for altered cellular responses. A cell cytotoxicity assay and MTT assay was used to determine the endothelial cell compatibility with X‐aerogel samples. Aggregometry and PAS results suggest that X‐aerogels of variable densities do not alter platelet functions. Endothelial cells were also compatible with X‐aerogel samples. Based on these results, we conclude that X‐aerogel is a viable biomaterial and future research will focus on determining the optimal density and porosity of X‐aerogel to maximize biocompatibility.
Modulation of Microvascular Smooth Muscle Adhesion and Mechanotransduction by Integrin‐linked Kinase
Authors: Sun Zhe , Huang Shaoxing , Li Zhaohui , Martinez‐Lemus Luis A. , Meininger Gerald A.
Published: June 2021

Integrin‐linked kinase (ILK) is a multi‐functional protein that acts both as a kinase and as a scaffolding protein in focal adhesions (FA). In this study we investigated the involvement of ILK in the adhesion of arteriolar vascular smooth muscle cells (VSMC) to fibronectin (FN) and in the mechano‐responsiveness of VSMC FA. ILK was knocked down in VSMC using ILK‐shRNA constructs. Atomic force microscopy (AFM) was used to characterize VSMC interactions with FN, to measure VSMC stiffness and to apply and measure forces at a VSMC single FA site. ILK was localized to FA and silencing ILK promoted cell spreading, enhanced cell adhesion, reduced cell proliferation, increased number of FAs and altered the expression of vinculin, paxillin and hic‐5. Silencing of ILK also enhanced α5β1 integrin adhesion to FN, decreased cell stiffness and increased contractile responses to application of a pulling force at a single FN‐FA site. The function of ILK in arteriolar VSMC appears linked to signaling pathways leading to inhibition of cell spreading, cell adhesion, FA formation, and mechano‐responsiveness. (Supported by NIH HL58690 to G.A. Meininger)
Gcn5 Functions in Mammalian Development
Authors: Atanassov Boyko , Hirsch Calley , Dent Sharon
Published: June 2021

We have created a series of mutations in the mouse Gcn5 gene in order to define the functions of this HAT in a mammalian system. Deletion of Gcn5 leads early embryonic death and to telomere dysfunction. Biochemical studies reveal that depletion of Gcn5 or ubiquitin specific protease 22 (Usp22), which is another bona fide component of the Gcn5‐containing SAGA‐type complex, increases the turnover of two shelterin proteins, TRF1 and POT1a. Inhibition of the proteasomes opposes this effect. The Usp22 deubiquitylating module requires association with SAGA complexes for activity, and we find that depletion of Gcn5 compromises this association in mammalian cells. Overall, these results indicate that Gcn5 regulates TRF1 levels through effects on Usp22 activity and SAGA integrity. These studies provide the first indication that Gcn5 and mammalian SAGA influence telomere maintenance and the first demonstration that SAGA affects protein stability. Current studies are directed at identifying additional USP22 targets.

We have also created Gcn5 null ES cells. Upon removal of LIF, these cells lose pluripotency more quickly than do wild type ES cells. Preliminary data suggest that Gcn5 may be a key regulator of the master transcription factors required for maintenance of the pluripotent state.
Molecular Mechanisms of Lysine Methylation
Authors: Trievel Raymond C , Del Rizzo Paul , Couture Jean‐Francois , Dirk Lynnette M. , Brunzelle Joseph S. , Houtz Robert L.
Published: June 2021

SET domain lysine methyltransferases (KMTs) are S‐adenosylmethionine (AdoMet)‐dependent enzymes that catalyze the site‐specific methylation of lysine residues in histones, transcription factors, and other protein substrates. SET domain KMTs also display product specificity, which is defined as their ability to catalyze different degrees of methylation of the lysine‐epsilon amine group, thus imparting an additional hierarchy in methyllysine signaling. To understand the mechanism underlying product specificity, we have characterized two active site mutants of the monomethylase SET7/9 that alter its specificity to a dimethylase and a trimethylase, respectively. Structures of the SET7/9 mutants in complex with peptides bearing unmodified, mono‐, di‐, and trimethylated lysines reveal that water molecules within the active site function as place holders that linearly align the lysine epsilon‐amine group with the methyl group of AdoMet to promote methyltransfer. As the methylation state of the lysine substrate increases during successive reactions, the water molecules dissociate from the active site, thereby enlarging the lysine binding channel to accommodate the increasing bulk of the methylated epsilon‐amine group. Collectively, our findings illuminate the catalytic roles of active site water molecules in facilitating lysine multiple methylation by SET domain KMTs.
Polycystin‐1 and ‐2 Dosage Regulates Pressure Sensing
Authors: Duprat Fabrice , Naeni Reza Sharif , Folgering Joost H.A. , Bichet Delphine , Lauritzen Inger , Malika Arhatte , Jodar Martine , Retailleau Kevin , Loufrani Laurent , Patel Amanda , Peters Dorien J.M. , Honoré Eric
Published: June 2021

Autosomal‐dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow‐sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch‐activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1‐deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.
Mild hyperhomocysteinemia increases atrioventricular nodal conduction: Role of the cardiac NMDA receptor
Authors: Maldonado Claudio J , Todnem Nathan D , Soni Chirag V , Givvimani Srikanth , Pushpakumar Sathnur B , Patibandla Phani K , Tyagi Suresh C
Published: June 2021

Hyperhomocysteinemia (HHcy) is a pathological condition resulting in elevated plasma concentrations of homocysteine (Hcy). Patients with HHcy have been reported to be at risk of sudden cardiac death (SCD). In previous electrophysiological (EP) studies in mice with HHcy, we observed an increase in atrioventricular (AV) nodal conduction rate. The mechanisms responsible for this alteration are unknown. Recent studies in mammals have shown that NMDA receptors (NMDARs) are present in heart, and Hcy is known to activate NMDARs. We hypothesize that in HHcy, cardiac NMDARs are hyperstimulated increasing both calcium transient currents in nodal cells and AV nodal conduction rate. To test this hypothesis two groups of C57BL/6 mice were treated with either Hcy in the drinking water or just water for 12 weeks. Standard EP studies were conducted using an octopolar catheter placed in the right ventricle. Surface and intracardiac ECGs were recoded and analyzed. A baseline EP study was conducted in all mice, followed by a second EP study, performed 30 min after IP administration of 20mg/Kg of magnesium sulfate (MgSO4) to block NMDARs. MgSO4 slowed AV nodal conduction in mice with HHcy but had no effect on control mice. These results suggest that acceleration of AV nodal conduction in HHcy may involve the cardiac NMDAR. Further studies using transgenic animals deficient in NMDAR are needed to confirm the role of this receptor.
Pan‐Histone deacetylase inhibition (HDACi) prevents heart failure with preserved ejection fraction (HFpEF) and cardiac fibrosis in high‐salt (HS) fed Dahl S rats (DS)
Authors: Plato Craig F , Perry Amy M , Joly Kristin M , Glascock Christopher , Cavasin Maria A
Published: June 2021

Chronic arterial hypertension (HYP) elicits cardiac hypertrophy and ultimately HFpEF, and >50% of patients diagnosed with heart failure have HFpEF. DS rats develop HYP, cardiac fibrosis and impaired cardiac diastolic performance in response to chronic HS feeding. HDACi is reported to decrease pathological cardiac hypertrophy, gene expression, and cardiac fibrosis. We evaluated the dose‐dependent effects of Scriptaid, a pan‐HDACi, to inhibit HFpEF and cardiac fibrosis in response to sustained HYP. DS were fed normal (0.49% NaCl) or HS (4.0% NaCl) and administered Scriptaid (3, 10, 30 mg/kg, i.p.) or vehicle (100% DMSO) daily for 6 wks. HS time‐dependently induced cardiac hypertrophy (endpoint: 26%) and impaired indices of diastolic performance (mitral Doppler E/A: −27% and isovolumic relaxation time: +30%) while not affecting left ventricular (LV) ejection fraction, and was associated with increased LV ANF and α‐skeletal actin mRNA expression and interstitial fibrosis (83%). Scriptaid dose‐dependently prevented reductions in diastolic performance and attenuated LV hypertrophy, interstitial fibrosis, and pathological gene expression despite not influencing severity of HYP. These results suggest HDACi may epigenetically modulate cardiac structural and functional remodeling responses to arterial hypertension thus improving cardiac performance in salt‐sensitive hypertension‐induced HFpEF.
Pulse Contour Analysis of Digital Arterial Pulse during Physical and Mental Stress Tests
Authors: Landin Sarah Elizabeth , Sorgert Kelly L , Esser Tom M , Klassen Michael C , Johannsen Anna C , Miller Andrew D , Hulsebus Minelle L , Johnson Bruce D , Baruch Martin C , Wilson Ted
Published: June 2021

Pulse Decomposition Analysis (PDA) offers new insights into factors altering the arterial pressure pulse contour. PDA premise is that peripheral arterial pressure pulse envelope consists of distinct component pressure pulses, a left ventricular ejection and its reflections. Two PDA parameters appear to be particularly useful in quantifying the pulse contour. P2P1 is the ratio of second component pulse and primary ejection pulse amplitudes. T13 is the temporal delay between the third component pulse and the primary ejection pulse. P2P1 and T13 parameters were used to examine pressure pulse contour changes in 81 healthy subjects (18–27 yrs; BMI 27.5 ± 2.6) undergoing a Stroop mental stress test, cycle ergometer stress tests (3 min; 50W and 100W), and post‐exercise recovery. Digital pressure pulse contours were recorded/quantified using PDA formalism. Relative to baseline, P2P1 increased 8% post‐Stroop (individual variation 0% to 15%), T13 increased 4%, (individual variation 0% to 8%). Exercise decreased P2P1 21 ± 14%, while the T13 change was 19 ± 15%. PDA‐based pulse propagation model quantifies physical changes that arterial pulse reflection sites undergo. Peripheral arterial pressure pulse appears to be due to the primary ejection pulse and two major reflections. Two reflection sites in the central arteries appear to be responsible for the “reflected” wave part of the pulse contour.
Effect of lateral decubitus position on circulation and heart rate variability in healthy adults
Authors: Sasaki Konosuke , Matsui Noriko , Nagai Mizuki , Haga Mayu , Maruyama Ryoko
Published: June 2021

There has been little research focus on the autonomic nervous system modulation in the lateral decubitus position. We compared the impact of the right and left lateral decubitus positions and the supine position on heart rate variability (HRV), including circulatory regulation. In 19 nonsmoking, healthy volunteers aged 24.8±4.2 years, we measured blood pressure (BP) in both upper arms and ECG. In addition, we assessed the condition of the inferior vena cava (IVC) by echocardiography in each posture. Subjects maintained each lateral decubitus position at an angle of about 45 degrees for 10 min at random after being in the supine position. Postural change was performed manually by two trained researchers. The systolic and diastolic BPs of the upper arm declined in both lateral positions. No significant differences were observed in high‐frequency or low frequency HRV or their ratio. However, in left lateral decubitus the HR was significantly lower than in the supine position throughout the experiment. In this position, the IVC diameter and area declined, although blood in the IVC flowed rapidly. The HRV findings suggest that the two recumbent positions have no influence on the autonomic nervous system. However, it is possible that the reduction in HR in left lateral decubitus position is followed by changes in the shape of the IVC.
Feeding resistant starch to Goto‐Kakizaki (GK) diabetic rats improves glucose status
Authors: Shen Li , Keenan Michael J , Zhou Jun , Martin Roy J
Published: June 2021

Dietary prebiotics have shown potential in the anti‐diabetes field. Our group has worked on resistant starch (RS), one type of prebiotic. We showed that dietary resistant starch possesses favorable impact on gut hormone profiles, including promoting GLP‐1 release consistently, an potent anti‐diabetic incretin (Zhou et al, 2006). Also we demonstrated dietary RS reduces body fat (Keenan, 2006; Shen, 2008). The current project is to determine if resistant starch can improve glucose status in a genetic animal model of diabetes. We hypothesize that dietary resistant starch can improve glucose metabolism in a type 2 diabetic rat model. The Goto‐Kakizaki (GK) rat is a non‐obese Wistar substrain which develops Type 2 diabetes mellitus early in life. In this study GK rats were fed a diet containing 30% resistant starch that was isocaloric and isonitrogenous when compared to the control diet. After 10 weeks of treatment, fasting blood glucose was reduced by feeding the resistant starch diet. It is proposed that altered gut fermentation and microbiota are the initial mechanisms by which RS improves diabetes and that enhance serum gut peptides (ie., GLP‐1, PYY and GIP) are secondary mechanisms involved in improve beta cell function and insulin sensitivity in this model of diabetes. This dietary approach is potentially of great therapeutic importance in the treatment of diabetes.
Deep Sequencing and Bioinformatics Analysis of Endothelial MicroRNA under Hypoxia Stress
Authors: Chen Zhen , Sun Wei , Padmanabhan Chellappan , Wang Wei‐Chi , Jin Hailing , Huang Hsien‐Da , Chien Shu , Shyy John Y‐J
Published: June 2021

We hypothesized that MicroRNAs (miRs) play an important role in endothelial responses to hypoxia. In order to identify miRNAs and to assess their expression patterns, small RNAs extracted from cultured human umbilical vein endothelial cells under normoxia or hypoxia (2% O2) were ligated with distinct RNA adaptors and underwent RT‐PCR to establish the cDNA library, which was then subjected to sequencing‐by‐synthesis deep sequencing, generating 106 small RNA sequences. Among 157 miRs identified, 41 were up‐ and 16 down‐regulated. Of interest, two highly conserved miR families were robustly induced by hypoxia, as validated by Northern blot. With the use of position weight matrix from TRANSFAC database and a prediction algorithm combining miRanda‐RNAhybrid‐TargetScan, both the transcription factors and the mRNA targets of these miRs were predicted. Hypoxia‐inducible factor 1α may target these miRs through hypoxia‐responsive elements located in the putative promoter region of these miRNA genes. As a consequence, these hypoxia‐induced miRs lead to the suppression of a panel of putative target genes, which were further verified by gain‐ or loss‐of‐function experiments using pre‐miRs or locked nucleic acids. By utilizing a strategy combining in silico and molecular and cellular experiments, we demonstrated that HIF1α can regulate endothelial miRs which in turn modulate downstream gene expression.
Phosphodiesterase‐5 Inhibition with Tadalafil Attenuates Left Ventricular Dysfunction and Cardiomyocyte Apoptosis in Doxorubicin‐induced Cardiotoxicity in Mice
Authors: Koka Saisudha , Das Anindita , Zhu Shu‐Guang , Durrant David , Xi Lei , Kukreja Rakesh C
Published: June 2021

Doxorubicin (DOX) is one of the most effective anticancer drugs. However, its cardiotoxicity remains a major clinical concern. As phosphodiesterase‐5 (PDE5) inhibitors are cardioprotective, we hypothesized that tadalafil (TAD), a long acting PDE5 inhibitor, would provide protection against DOX‐induced cardiotoxicity. CF‐1 mice received Saline (0.2 ml ip), DOX (15 mg/kg ip) or DOX+TAD (4 mg/kg po for 9 days; n=18–24/group). Left ventricular (LV) function was assessed by echocardiography and Millar catheter. DOX‐mediated impairment of LV function was significantly reduced by TAD+DOX (p<0.05). Western Blots revealed that DOX‐mediated reduction of Bcl‐2 and GATA‐4 were maintained by TAD+DOX. Antioxidant capacity was also improved through enhanced MnSOD in TAD+DOX. Immunofluorescence staining of Desmin and TUNEL assay showed that DOX‐induced myofibrillar disarray and apoptosis were attenuated by TAD. TAD+DOX significantly increased cGMP level and PKG activity in heart as compared to DOX. Co‐treatment of TAD with DOX did not interfere with the anti‐cancer efficacy of DOX, as determined by human osteosarcoma (OSA‐1) cell viability assay and tumor xenograft model. In conclusion, TAD improved LV function and prevented cardiomyocyte apoptosis via upregulation of cGMP, PKG and MnSOD, without interfering with the chemotherapeutic benefits of DOX. (Supported by NIH HL51045, HL79424, HL93685 & AHA 0765273U)
Protective effect of baicalein on cardiomyoblasts H9C2 against damage induced by oxidative stress
Authors: You Ya‐Ching , Ou Hsiu‐Chung
Published: June 2021

Oxidative stress plays an important role in many cardiovascular diseases. In this study, the effects of baicalein, a natural flavonoid isolated from the root of Scutellaria baicalensis G., on hydrogen peroxide (H2O2)‐induced cytotoxicity in cardiomyoblasts H9C2 were investigated. Treatment with H2O2 markedly decreased the viability of H9C2 cells in a dose‐ and time‐dependent manner, accompanied by increased reactive oxygen species (ROS) generation, mitochondrial dysfunction, downregulation of Bcl‐2, upregulation of Bax, and subsequent cell apoptosis. Pretreatment of H9C2 cells with baicalein inhibited H2O2‐induced cell viability loss, intracellular ROS generation. Furthermore, baicalein potentially inhibited H2O2‐induced cell apoptosis characterized with the DNA fragment. Consistently, the mitochondrial pathway involving the mitochondrial dysfunction associated with cell apoptosis including membrane potential loss, the release of cytochrome c, the disturbance of Bcl‐2 family proteins induced by H2O2 were also abrogated in the presence of baicalein. Taken together, these results suggest that baicalein can block H2O2‐induced apoptosis by prevention of oxidative stress as well as regulation of Bcl‐2 family members and suppression of mitochondria dysfunction, which might be beneficial for the treatment of oxidative stress‐associated cardiovascular diseases.
Ankle Blood Pressure: A Novel Measure Affecting Central Arterial Wave Reflection?
Authors: Tarumi Takashi , Sugawara Jun , Tanaka Hirofumi
Published: June 2021

Objective

There is an increasing recognition that central aortic pressure is more relevant than peripheral (brachial) measure for the prediction and pathophysiology of cardiovascular disease. Central pressure is greatly influenced by the phenomenon of arterial wave reflections returning from the peripheral vasculature, which can be quantified by augmentation index. Primary wave reflections returning to the heart are thought to come from the lower body (e.g., lower limbs). Accordingly, the primary aim of the present study was to determine the association between central augmentation index and arterial blood pressures recorded in both the upper and lower limbs.

Methods

A total of 1,189 apparently healthy adults (567 males and 622 females) of varying ages were studied. Brachial and ankle blood pressure, carotid augmentation index, and brachial‐ankle pulse wave velocity were measured.

Results

All of the blood pressure (brachial and ankle, systolic, mean, and diastolic) measurements were significantly associated with carotid augmentation index. Among them, ankle mean arterial pressure was the strongest correlate of carotid augmentation index (r=0.56, P<0.05). This relation remained highly significant even after the influence of brachial blood pressure or age was accounted for in the partial correlation analyses. Moreover, stepwise regression analyses revealed that ankle mean arterial pressure was the strongest independent predictor of carotid augmentation index among all of the blood pressure measurements.

Conclusion

Ankle blood pressure is strongly associated with the augmentation of central blood pressure, and this relation is independent of other blood pressure measures (brachial blood pressure).
Circulating angiogenic factors in a rat model of preeclampsia
Authors: Agunanne Enoch , Uddin Mohammad N , Horvat Darijana , Puschett Jules
Published: June 2021

Objective

We studied the angiogenic factors in our rat model of preeclampsia (PE) in which marinobufagenin (MBG, a bufodienolide) plays an important role.

Methods

Animals were grouped into: normal, non‐ pregnant (C); normal pregnant (NP); NP injected weekly with DOCA intraperitoneally (IP) and whose drinking water was replaced with 0.9% saline (PDS); NP given daily injections of MBG (0.765 μg/100g) once pregnancy was established; PDS given daily injections of resibufogenin [RBG, an antagonist of MBG (30 μg/kg/day)] IP from day 4 of pregnancy (PDSR). The angiogenic factors were measured on 3–5, 7–10 & 17–20 days of gestation. We have previously shown that MBG levels are already elevated in this model at the 3–5 day time period and remain elevated throughout gestation in PDS rats.

Summary of Results

At 3–5 days of pregnancy, there was no angiogenic imbalance. At the 7–10 day period, plasma PlGF was greater in the NP than in the PDS. The sFlt‐1/PlGF as well as the placental Flt‐1 and Flt‐1/PlGF were greater in the PDS rats than in NP (p<0.05). These findings were also present at the 17–20 day time point. The levels of these factors in PDSR were similar to those in NP.

Conclusions

Angiogenic imbalance contributes to the pathogenesis of the preeclamptic syndrome in our rat model. MBG elevation precedes this alteration in angiogenic factors. This is confirmed by prevention of this imbalance in this study by pretreatment with RBG. Scott, Sherwood/Brindley Foundation.
2‐Methoxy Estradiol Mediated Downregulation of Angiotensin Type 1 Receptor Expression
Authors: Koganti Sivaramakrishna , Thekkumkara Thomas
Published: June 2021

Cardiovascular disease (CVD) is the leading cause of death among women in the United States yet develops on average 10–15 years later than in men. This delayed onset could be in part though not well understood, due to the protective effect of estrogen before menopause. Experimental studies have identified angiotensin type 1 receptor (AT1R) as a key factor in the progression of CVD. In this study, we examined the effects of estrogen metabolite 2‐Methoxy Estradiol (2ME2) on AT1R expression. Rat liver cells were exposed to 2ME2 for 24 h and angiotensin II (AngII) binding and AT1R mRNA expressions were assessed. In the presence of 2ME2, cells exhibited significant downregulation of AngII binding in a dose and time dependent manner. Downregulation of Ang II binding was AT1R specific with no change in receptor affinity. Under similar conditions, we observed inhibition of the AT1R mRNA and increased phosphorylation of both MAP‐kinases (MAPK) p42 and p44. Pretreatment of cells with MAPK specific inhibitor prevented 2ME2 induced MAPK phosphorylation and downregulation of the AT1R suggesting that the observed inhibitory effect is mediated through MAPK signaling intermediate(s). Since AT1R plays a critical role in the control of hypertension and other cardiovascular diseases, 2ME2‐mediated changes in receptor expression can further enlighten the importance of estrogen. (Supported by NIH Grant DK072140)
Chronic placental ischemia decreases insulin‐like growth factor‐1 during late gestation in the rat
Authors: Bozadjieva Nadejda Ivanova , Soldner Emma L B , Heltemes Alaina , Gingery Anne , Gilbert Jeffrey S
Published: June 2021

Preeclampsia and other pregnancy related hypertensive disorders affect ~ 8 % of all pregnancies and are a major source of maternal and neonatal morbidity and mortality. Although previous research indicates insulin‐like growth factor‐1 (IGF‐1) and its binding proteins (IGFBP) are important in preeclampsia and intrauterine growth restriction (IUGR), whether they are a cause or consequence of placental ischemia is unclear. We hypothesized that chronic placental ischemia during late pregnancy in the rat would reduce maternal levels of circulating IGF‐1 and IGFBP‐3. Rats were assigned to either reduced uteroplacental perfusion (RUPP; n=15) or normal pregnant control (NP; n=12) groups. RUPP was induced by placing silver clips on the abdominal aorta and ovarian arteries on day 14 of gestation. On day 19 of pregnancy, arterial pressure (124±4 vs. 106±2 mmHg; P<0.05) was increased in the RUPP vs. NP dams. Fetal (2.3±0.03 vs. 2.5±0.04g; P<0.05) and placental (0.42±0.01 vs. 0.46±0.01g; P<0.05) weights, plasma IGF‐1 concentrations (271.7 ± 16.1 vs. 325.6 ± 21.3 ng/mL; P<0.05) and serum IGFBP‐3 levels (0.12±0.07 vs. 0.29±0.02; P<0.05) were decreased in the RUPP vs. NP dams. These data identify an association between placental ischemia and the IGF system during pregnancy and support the hypothesis that placental ischemia results in decreased IGF‐1 and IGFBP‐3. Further, these data suggest that in addition to IUGR, IGF‐1 may play a role in placental ischemia‐induced hypertension.
Chronic treatment with an orally active angiotensin‐(1‐7) formulation decreases arterial pressure, cardiac hypertrophy and improves cardiac function in spontaneously hypertensive rats
Authors: Bertagnolli Mariane , Casali Karina Rabello , Pinto Graziela , Dias Lucinara Dadda , Rigatto Katya , Schaan Beatriz D , Irigoyen Maria Cláudia , Santos Robson A S
Published: June 2021

Considering that ANG‐(1‐7) counterbalances angiotensin II hypertensive and hypertrophic effects, we aimed to assess whether chronic treatment with ANG‐(1‐7) modifies arterial blood pressure (BP), cardiac hypertrophy (CH), and cardiac function in spontaneously hypertensive rats (SHR). Male SHR (15 weeks) were treated with tap water (n=9) or oral administration of an ANG‐(1‐7)‐cyclodextrin inclusion compound (corresponding to 30mg/Kg/day Ang‐(1‐7), n=8) by gavage (10 weeks). BP (mmHg) was assessed by intraarterial catheterization, and the left ventricle (LV) pressures through carotid catheterization. CH index (CHI) was estimated by heart/body weight ratio. Data are presented as mean±SD. ANG‐(1‐7) reduced diastolic (147±5 vs 162±7), systolic (222±7 vs 202±7), and mean arterial pressures (191±6 vs 178±7). The end‐diastolic (17±5 vs 10±2) and the systolic (247±35 vs 216±34) LV pressures were decreased after treatment. Chronic ANG‐(1‐7) also slightly decreased CHI (4.12±0.10 vs 3.90±0.15) in SHR. These results indicate that chronic ANG‐1‐7) treatment decreased BP and improved cardiac function in SHR with established hypertension. The data reinforce the potential pharmacological use of ANG‐(1‐7) in managing cardiovascular diseases. Supported by: INCT‐Nanobiofar, CNPq, CAPES.
Differences in Lung and Right Ventricle Responses to Sustained and Intermittent Hypoxia
Authors: Joy Anne Marie , Zhang Jianhua , Dai Qiuxia , Mifflin Steve W. , Lindsey Merry L.
Published: June 2021

Sustained hypoxia (SH) induces pulmonary arterial hypertension, a model of right heart failure. Intermittent Hypoxia (IH) induces both pulmonary and systemic arterial hypertension and is a model of arterial hypoxemia accompanying occlusive sleep apnea. Our long‐term goal is to capitalize on differences between these two models to dissect out the pathophysiological mechanisms that regulate lung and heart function. We hypothesized that sustained and intermittent hypoxia would differentially alter lung and heart structure and function. We collected left ventricle (LV), right ventricle (RV), and lung tissue from three groups of rats subjected to 7 days of a) SH (n=12); b) IH (n=12); and c) normoxic controls (n=18). The lung to body weight ratios were increased in the SH group compared to the normoxic and IH groups (7.03±0.31 mg/g vs 4.72±0.14 mg/g and 4.74±0.16 mg/g, respectively (p<0.05 for SH compared to both normoxic and IH groups). The RV to body ratios were also increased in the SH group compared to the normoxic and IH groups (0.47±0.01 mg/g vs 0.32±0.01 mg/g and 0.36±0.01 mg/g, respectively (p <0.05 for SH compared to both normoxic and IH groups). The increase in the lung and RV masses indicates fluid accumulation in these organs, consistent with pulmonary hypertension and right heart dysfunction. The LV to body weight ratios were similar among the groups (2.35±0.07 mg/g for the SH; 2.29±0.03 mg/g for the normoxia, and 2.22±0.04 mg/g for the IH; p=ns). Our results show that the two models display different responses in terms of increases in organ weights. We can now use these tissues to explore changes in extracellular matrix composition to determine if early gene changes can be delineated in the two types of hypoxia.