bluuebunny/crossref_metadata_2025 huggingface dataset:
PECAM‐1 Gene Ablation Impairs Endothelium‐Dependent Relaxation
Authors: Sutliff Roy L , El‐Ali Alexander M , DeLisser Horace M , Hart C. Michael
Published: June 2021

Platelet endothelial cell adhesion molecule‐1 (PECAM‐1 or CD31), a 130 kDa type I glycoprotein, is a complex molecule that has been widely implicated in the pathogenesis of cardiovascular conditions. Current evidence indicates this molecule serves as a mechanosensor that is integral to flow mediated vascular remodeling. We hypothesize that PECAM‐1 may regulate vascular function through the NO signaling pathway. Contractile responses to the receptor‐independent agonist, KCl, and the receptor‐dependent agonist, phenylephrine, were measured in aortic rings from wild‐type (WT) and PECAM‐1 knockout (KO) mice. Force generation was analyzed with respect to agonist sensitivity and force/cross‐sectional area. Relaxation responses to the endothelium‐dependent agonist, acetylcholine, and the endothelium‐independent agonist, sodium nitroprusside (SNP), were also measured. Contractile sensitivity and maximum force to phenylephrine or KCl was comparable in WT and KO rings. Although aortic rings from WT and KO mice responded similarly to SNP, rings from KO mice were less sensitive to acetylcholine‐mediated relaxation (p<0.01; n=5). These findings indicate that PECAM‐1 plays a significant role in acetylcholine‐mediated vascular relaxation and suggest that alterations in PECAM expression or function may contribute to vascular dysregulation. Supported by HL070892 (RLS); HL079090 (HMD) and the Atlanta VA Research Service (CMH)
Luminal endothelial lectins with affinity for N‐Acetylgucosamine determine flow‐induced cardiac and vascular paracrine responses
Authors: Rubio Rafael , Ramiro‐Diaz Juan , Barajas‐Espinosa Alma , Chi‐Ahumada Erika , Perez‐Aguilar Sandra , Torres‐Tirado David , Castillo‐Gonzalez Jesus , Knabb Maureen , de la Rosa Ana Barba
Published: June 2021

Coronary endothelial luminal membrane (CELM) oligosaccharides (O) and lectins (L) are involved in flow detection. Flow may induce a reversible L‐O interaction causing flow‐induced endothelial (FIE) cardiac effects. Because N‐Acetylglucosamine (GlcNac) is a component of glycocalyx O, we isolated CELM GlcNac‐binding L (using GlcNac‐affinity probe) and show their role in cardiac and vascular FIE responses. The GlcNac‐affinity probe is a 460 kDa GlcNac polymer (GlcNac‐Pol). In the heart: 1) Intracoronary given GlcNac‐Pol upon binding to CELM, reduces the FIE inotropic and dromotropic effects. 2) GlcNac‐Pol used as an affinity probe isolated CELM GlcNac‐recognizing L; 35 individual L were identified, some of which likely participate in FIE and in GlcNac‐Pol‐induced effects. 3) In isolated blood vessels perfuse at controlled flow rates; FIE vasodilatation (FIEV) is blocked by: binding GlcNac‐Pol to CELM, Hyaluronidase, L‐NAME plus Indomethacin and Amiloride; a ENaC blocker. We also show ENac is present in CELM and is a L. These suggest FIEV is due to: a) FIE ENaC‐hyaluronidate (a GlcNac polymer) interaction, b) release of NO and prostaglandins and c) GlcNac‐Pol upon binding to ENaC inhibits release of paracrine mediators. Funded by CONACyT SEP‐42567, CONACyT‐SALUD 2004‐C01‐156.
Biochemical alterations in coronary endothelial luminal membranes during ischemia and reperfusion in isolated perfused guinea pig hearts
Authors: Knabb Maureen T , Pérez‐Aguilar Sandra , Torres‐Tirado David , Ramiro‐Díaz Juan , Rubio Rafael
Published: June 2021

Ischemia/reperfusion has marked effects on the thickness of the coronary endothelial glycocalyx but little is known about the biochemical alterations associated with this structural change. Lectinic and glycan‐containing molecules have been implicated in flow‐induced changes in myocardial conduction and contractility but the mechanism is unknown. In order to identify the effects of ischemia and reperfusion on endothelial flow‐specific biomarkers, isolated perfused guinea pig hearts were treated with ischemia followed by reperfusion. Luminal coronary endothelial membranes were isolated via the silica pellicle method in control, ischemic, or ischemia/reperfusion treated hearts. Flow sensitive proteins including PECAM, VCAM, ENaC, B2, AT1 and AT2 receptors were decreased in luminal membranes in reperfused as compared to ischemic hearts. Anionic glycoconjugates (GAGs) quantified with Alcian blue on PVDF membrane decreased from 0.95 to 0.35 mg GAG/mg protein in ischemic hearts but showed partial recovery with reperfusion to 0.52 mg GAG/mg protein. These results indicate that ischemia leads to loss of glycosylated proteins that are partially restored with reperfusion. However, flow sensitive proteins are lost from the endothelium, which may explain the deleterious effects of reperfusion following ischemia. Funded by CONACyT SEP‐42567, CONACyT‐SALUD 2004‐C01‐156, Fulbright‐ Garcia Robles #9583.
Tissue engineered cardiac stem cell grafts for repairing heart with myocardial infarction
Authors: Kim Deok‐Ho , Smith Rachel , Gupta Kshitiz , Kim Pilnam , Suh Kahp‐Yang , Marban Eduardo , Levchenko Andre
Published: June 2021

Recently, putative adult cardiac stems cells have been isolated and investigated in the laboratory and clinical settings. In spite of demonstrated repair potential, the utility of transplantation of these cells remains in doubt due to high cell mortality and low tissue integration. Here we demonstrate that the scalable (>1 cm2) nanostructured scaffold of biocompatible hydrogels can produce structurally aligned cardiac stem cell sheets, which mimic the aligned cardiophysiological architecture seen in normal heart tissue in vivo. Through in vitro characterization, we also demonstrate that topographically‐controlled cardiac stem cells grown on the scaffolds show significant increase in cell adhesion, migration, and proliferation compared to the control group. Furthermore, in vitro co‐culture of neonatal rat ventricular myocytes with lentivirally transduced cardiac stem cells expressing NCX promoter‐GFP exhibited enhanced cardiac differentiation on nanofabricated scaffolds. To test the therapeutic potential in vivo, we engrafted the monolayered cardiac stem cell patches onto the infarcted heart of adult rats. We found that engrafted GFP‐labelled cardiac stem cells migrated into and integrated with host cardiac tissue. This was evidenced by formation of stable gap junctions and expression of the cardiac specific marker Troponin I. There was a clear improvement in tissue repair as indicated by thicker infarct walls in those animals that received the stem cell graft. Overall, we argue that our method not only enables development of functional cardiac grafts, but also provides a novel therapeutic approach to promote cardiac regeneration and improve healing in a rat myocardial infarction model.
Sex‐based Differences in Ventricular Collagen Profile of Spontaneously Hypertensive Rats
Authors: Krontiris‐Litowitz Johanna , Fulton Benjamin
Published: June 2021

In this project we investigate differences in myocardial collagen by comparing the collagen profile of hypertrophied ventricles of male and female Spontaneously Hypertensive Rats (SHR). Ventricular tissue harvested from 12 month male and female SHR (n=7,7) was homogenized in PBS and SDS, centrifuged at 15000 rpm, resuspended in 7% Formic Acid and digested with CnBr. The resultant peptide fragments in this acid soluble fraction were separated by 2D gel electrophoresis and analyzed using PDQuest software. The protein content in the acid soluble fraction of males was significantly greater than females (95 ug+−7, 53.3 ug+−2; p<.05). The peptide profile of males differed from females. A total of 25 peptide fragments were identified in male ventricles while only 21 fragments were identified in the equivalent fraction (70 ug protein) of female ventricles. Seven of the fragments identified in males were not visible in female tissue. The collagen profile of males differed from females. The concentration of one of the peptide fragments characteristic of collagen III was 5 fold less in female hearts than in male hearts. Additionally, two peptides characteristic of collagen I and a third, characteristic of collagen III, were not visible in female ventricles. This data suggests that the architecture of the hypertrophied heart and more specifically the collagen profile may not be the same in the males and females.
Endothelial beta1 integrins regulate sprouting and network formation during vascular development
Authors: Wenzel Daniela , Malan Daniela , Schmidt Annette , Geisen Caroline , Raible Axel , Bölck Birgit , Fleischmann Bernd K. , Bloch Wilhelm
Published: June 2021

Beta1 integrins are important regulators of vascular differentiation and development since their endothelial‐specific deletion results in embryonic lethality. In the present study we have investigated the molecular mechanisms underlying the prominent vascular abnormalities in the absence of beta1 integrins. Because of the early embryonic lethality of knock out mice we have studied endothelial cell and vessel development in beta1 integrin deficient murine embryonic stem (ES) cells. We found that vessel development was strongly defective in the mutant embryoid bodies (EBs), as only primitive and short sprouts developed from clusters of vascular precursors in beta1 integrin (−/−) EBs whereas complex network formation of endothelial tubes was observed in wild‐type EBs. The vascular defect was due to deficient beta1 integrin expression in endothelial cells as endothelial‐specific re‐expression rescued the phenotype entirely. The mechanism responsible for defective vessel formation was reduced endothelial cell migration and elongation. Moreover, the apoptosis vs proliferation rate increased during differentiation resulting in a reduction of endothelial cells. This effect was due to an elevation of peNOS and pAKT signaling molecules, respectively. Thus, endothelial beta1 integrins are determinants of vessel formation and this is mediated via different signaling pathways.
Inhibition of Matrix Metalloproteinase‐9 (MMP‐9) Reverses Changes in Vascular Wall Structure and Function of Thoracic Aorta of Dahl Salt‐Sensitive (DSS) Rats
Authors: Rodriguez‐Alvarez Walter E , Tyagi Neetu , Sen Utpal , Adeagbo Ayotunde S , Joshua Irving G , Tyagi Suresh C
Published: June 2021

Left ventricular hypertrophy and cardiac fibrosis in DSS‐hypertensive rats were associated with increased MMP‐9 and lowered TIMP‐4. The objective of this study was to determine if salt sensitive hypertension induced changes in vascular structure and function were ameliorated by inhibition of MMP‐9. Vascular function (isolated aortic rings) and levels of MMP‐9 and TIMP‐4 (Western blot, reverse zymography, and confocal staining) were determined in thoracic aorta segments of groups of 16 week male DSS, and Lewis control (L) rats on a normal salt or a high salt diet (8 wks). Rats were regrouped into DSS plus MMP‐9 inhibitor (SB‐3CT) treatment (D+I), and DSS without inhibitor (D). Acetylcholine‐induced relaxation responses were decreased in D as compared to L, but normalized in D+I. The contraction responses to phenylephrine increased by 20% in aortic rings from D as compared to L, but normalized in D+I. The sodium nitroprusside‐induced relaxation responses in all groups were not altered. The levels of MMP‐9 were higher in aortic segments from D as compared to the L, and were significantly reduced in D+I to levels comparable to those recorded for the L group. The levels of TIMP‐4 were lower in vessel segments from D as compared to L, but normalized in D+I. The results suggested that increase MMP‐9 instigated vascular hypertrophy and dysfunction in Dahl salt‐sensitive rats.
The role of P2Y2 receptor activation in pro‐fibrotic responses of cardiac fibroblasts
Authors: Lu David , Braun Oscar Ö. , Aroonsakool Nakon , Insel Paul A.
Published: June 2021

Cardiac fibroblasts (CFs) play an essential structural role in the heart and in cardiac remodeling after injury. CFs are activated and adopt a pro‐fibrotic phenotype in response to a variety of growth factors including extracellular nucleotides. Such nucleotides can interact with 8 known P2Y receptors, G‐protein‐coupled receptors (GPCRs); P2Y2 receptors (P2Y2R), which are activated by ATP and UTP, are the most abundant P2Y receptor expressed by rat cardiac fibroblasts. In this study we examined the effect of UTP on fibroblast transformation. UTP dose‐dependently increased the expression of pro‐fibrotic markers, including plasminogen activator inhibitor‐1, transforming growth factor‐ß and α‐smooth muscle actin. In addition, UTP increased proliferation and migration of CFs. Effects similar to those induced by UTP occurred with the non‐hydrolyzable analog UTPγS. The stimulatory effects of UTP on pro‐fibrotic gene expression and CF proliferation did not occur in CF from P2Y2R‐knockout mice. Thus, UTP promotes CF transformation to a pro‐fibrotic phenotype through activation of P2Y2R. Our results suggest that UTP and other extracellular nucleotides (e.g. ATP) may act via P2Y2R to regulate CF activation and migration during normal and injury‐induced cardiac remodeling.

Supported by grants from NIH (GM007752, HL066941, GM066232) and the Ellison Medical Fdn.
Fibroblasts migrate faster in the direction of applied tension in a collagen‐fibrin wound model
Authors: Rouillard Andrew D. , Holmes Jeffrey W.
Published: June 2021

After myocardial infarction, the structural and mechanical features of replacement scar tissue, particularly the degree of collagen fiber alignment, are an important determinant of ventricular function. We have found that in healing infarcts, collagen becomes aligned in the direction of greatest stretch, but the mechanism is not yet known. The aim of this work was to test whether cardiac fibroblasts migrate into a wound faster in the direction of tension, which may explain subsequent collagen alignment. Fibroblast‐populated collagen gels were cast in square molds. An incision made in the center of each gel was filled with fibrin to simulate a wound. Tension was applied either transversely or axially with respect to the long axis of the incision. After two days of culture, cell nuclei were fluorescently imaged. The distribution of cells migrating transversely into the incision was fit to the diffusion equation. The ratio of diffusion coefficients showed migration was 3.1±0.64 times faster when cells were migrating parallel, rather than perpendicular, to the direction of tension (p = 0.03). We conclude that tension can bias cardiac fibroblast migration, and that this effect merits further investigation as a potential mechanism for collagen alignment in scar tissue. This work was funded by the American Heart Association Pre‐doctoral Fellowship Program.
IL‐1β and TNF‐α Differentially Regulate Lysyl Oxidase Expression and Activity in Adult Cardiac Fibroblasts
Authors: Voloshenyuk Tetyana G , Gardner Jason D
Published: June 2021

Cardiac fibroblasts constitutively express lysyl oxidase (LOX), an enzyme that catalyzes the crosslinking of collagen and modulates extracellular matrix maturation. Pro‐inflammatory cytokines, IL‐1β and TNF‐α, are elevated during cardiac disease and are known to modulate LOX expression in non‐cardiac cells. However, little is known about the regulation of LOX expression or activity in the heart. We tested the hypothesis that IL‐1β and TNF‐α decrease LOX expression and activity in isolated adult rat cardiac fibroblasts. After incubation in minimal serum media, fibroblasts were treated with TNF‐α (20 ng/ml) or IL‐1β (10 ng/ml) for 48 hrs. Cells were harvested and LOX expression assayed by Western blot. LOX activity was measured in cell culture media using Amplex red assay. Treatment with TNF‐α led to a significant reduction in LOX protein expression. However, IL‐1β did not affect LOX expression. Despite these differences in expression, both TNF‐α and IL‐1β significantly decreased LOX activity (64% and 60% vs. control, respectively). In addition, the TNF‐α regulation of LOX protein occurs in part through a Rho‐associated protein kinase (ROCK) pathway, as incubation of fibroblasts with the ROCK inhibitor, Y‐27632, attenuated the reduction of LOX expression. These findings indicate that the expression and activation of LOX in cardiac fibroblasts is differentially regulated by TNF‐α and IL‐1β.
Progress towards regenerating the corticospinal tract after spinal cord injury
Authors: Steward Oswald
Published: June 2021

Most of the symptoms resulting from spinal cord injury are due to the interruption of the long tracts that interconnect the brain and spinal cord. Thus, promoting regeneration of these tracts is considered a clinically relevant approach to enhancing recovery of function in patients with spinal cord in¬jury. Finding a way to induce regeneration of the cortico‐spinal tract (CST) is of particular importance because this pathway mediates voluntary movement. Even small increments in function could be of great importance for people who have suffered SCI. Here, I will describe our studies that seek to develop interventions to promote the regeneration of CST axons in mice and rats in ways that may be translatable to the clinic. The approach uses genetically modified mice in proof of concept experiments to identify key molecular pathways followed by interventional approaches using viral vectors and small molecules to target the same pathways. I will describe the limited regenerative growth of the CST that occurs in normal rodents and the degree to which regenerative growth can be enhanced. A key question yet to be resolved whether the regenerative growth of CST axons that can be achieved is capable of mediating recovery of motor function.
A Fibronectin Fragment Elicits Vasodilatation and Alters Myogenic Responsiveness of Skeletal Muscle Arterioles
Authors: Chao J‐T. Sherry , Davis George E , Davis Michael J
Published: June 2021

Interactions between integrin‐extracellular matrix proteins are important modulators of vascular tone that are altered during vascular remodeling, wound healing, and inflammation. Degraded collagen, which contains integrin‐binding sequences Arg‐Gly‐Asp (RGD), has been shown to modulate vascular tone by altering calcium entry. In this study, we investigated the effects of a proteolytic fragment of fibronectin (FN120) on vascular tone in isolated, cannulated, and pressurized rat skeletal muscle arterioles. Abluminal incubation with FN120 (1, 10, 100 μM) for 15 minutes elicited a dose‐dependent, transient dilatation at 90 cmH2O intraluminal pressure, with 10 μM FN120 producing significant dilation (n=9, p=0.03). FN120 (10 μM) also significantly increased the steady‐state myogenic index (−0.3; control: −0.13; n=9, p=0.02), as measured over the pressure range of 60–180 cmH2O. After wash‐off and re‐application of FN120, the time to peak transient dilation at 60 cmH2O was markedly shortened (from 8 to 1.5 minutes for 10 μM FN120), compared to the first application. To determine if these effects depended on FN‐integrin interactions, arterioles were pre‐incubated with a β1 integrin function blocking antibody (26 μg/mL) prior to addition of 10 μM FN120. The β1 integrin antibody blunted the steady‐state dilation to FN120 (25% decrease; p=0.039) and attenuated the increased myogenic index (−0.3 to −0.1; n=3, p=0.03). Our data suggest that the interaction between this FN fragment and β1 integrin may be important components of vascular remodeling. Supported by NIH 072989 to MJD.
DIABETES DOES NOT ALTER CONTRACTILE FUNCTION IN HIBERNATING MYOCARDIUM OF CONSCIOUS SWINE
Authors: Sharma Sandeep , Yoon Sung‐Ho , Cook Jeffrey , Saad Maged M. , Eom Woosik , Crystal George J. , Kim Song‐Jung
Published: June 2021

It has been proposed that a shift to glucose metabolism is integral to development of hibernating myocardium (HM). We tested the hypothesis that diabetic hearts have impaired contractile function during hibernation. Fifteen swine were divided into control (CON; n=8) and streptozotocin (STZ)‐treated (100 mg/kg; n=7) groups. Animals were chronically instrumented to measure coronary blood flow (CBF) and regional wall thickening (WT). Aortic and coronary sinus blood samples were obtained so that myocardial glucose uptake (MGU) could be calculated. HM was induced by 6 repetitive episodes of 90‐min coronary stenosis (30% reduction in baseline CBF) followed by reperfusion every 12 h. Inotropic reserve was assessed with isoproterenol (ISO, 0.2 μg/kg/min, i.v.). Plasma glucose was elevated in STZ compared to CON. In CON, MGU was increased 2‐fold during hibernation compared to baseline, whereas MGU was not affected in STZ; glycogen deposition was prominent in CON but negligible in STZ. Nevertheless, decreases in WT in HM stabilized at a similar level in CON and STZ (−22% and −14%, respectively). Furthermore, ISO caused equivalent, substantial increases in WT in HM of CON and STZ. We conclude that 1) diabetes does not impair contractile function in HM, 2) augmented glucose metabolism is not an essential feature of HM, and 3) inotropic reserve of HM remains intact in diabetic hearts.
Mitigation of Heart Failure Progression with Sildenafil Involves Inhibition of RhoA/Rho‐Kinase Pathway
Authors: Salloum Fadi N , Chau Vinh Q , Hoke Nicholas N , Abbate Antonio , Kukreja Rakesh C
Published: June 2021

Chronic inhibition of phosphodiesterase‐5 (PDE‐5) with sildenafil after permanent occlusion of the left anterior descending coronary artery (LAD) limits ischemic heart failure (HF) in mice. We postulated that treatment with sildenafil beginning at 3 days post myocardial infarction (MI) would also attenuate HF through inhibition of RhoA/Rho kinase pathway.

Adult male ICR mice with fractional shortening (FS) less than 25% at day 3 following permanent LAD ligation were treated with either saline (volume matched, ip, BID) or sildenafil (21 mg/kg, ip, BID) for 25 days. Echocardiography showed FS preservation and less left ventricular dilatation with sildenafil treatment versus saline at 7 and 28 days post‐MI (P<0.05). Both fibrosis and apoptosis, determined by Masson's trichrome and TUNEL, respectively, were attenuated with sildenafil (P<0.05 vs. saline). Western blotting showed enhanced Bcl‐2/Bax ratio with sildenafil (P<0.05 vs. saline). Activity assay showed sildenafil‐mediated PKG activation 1 day after treatment (P<0.05 vs. sham and saline). Activation of PKG with sildenafil was associated with inhibition of Rho kinase (P<0.05), as compared to saline.

Our findings show that chronic sildenafil treatment in mice with HF attenuates LV dysfunction independent of its infarct‐sparing effect by inhibiting the RhoA/Rho kinase pathway. This research was funded by NIH (HL51045, 79424 and 93685 to RCK).
High dietary sugar exacerbates cardiac reperfusion injury in perinatal taurine depleted adult rats
Authors: Roysommuti Sanya , Kulthinee Supaporn , Jirakulsomchok Dusit , Wyss J. Michael
Published: June 2021

Reperfusion injury triggers acute and chronic damage to multiple organs. Taurine treatment appears to reduce these injuries. This study tests the hypothesis that perinatal taurine exposure reduces the severity of cardiac reperfusion injury in the adult male rats. Female Sprague‐Dawley rats were fed normal rat chow with 3% beta‐alanine (taurine depletion, TD), 3% taurine (taurine supplementation, TS) or water alone (control, C) from conception to weaning. Male offspring were fed normal rat chow and water containing 5% glucose (CG, TSG, TDG) or water alone (CW, TSW, TDW) throughout the experiment. At 7–8 weeks of age, all rats were anesthetized and their trachea clamped until cardiac arrest occurred and mean arterial pressure fell below 60 mm Hg. The clamp was immediately released and cardiopulmonary resuscitation was performed with cardiac function returning within 4 min. Circulating markers of cardiac injury were measured 2 days later. Plasma aspartate aminotransferase increased in all glucose treated and TDW rats, but the increase was nearly 3X greater in TDG vs. any other group. Creatine kinase‐MB increased in all treatment groups but was far greater in TD than other groups. Troponin‐I and brain natriuretic peptide were greatly increased in TDG compared to all other groups. These data indicate that high dietary sugar together with early life taurine depletion exacerbates cardiac reperfusion damage in adult rats.
Palmitate evokes ceramide‐dependent reactive oxygen species (ROS) generation from sources other than NADPH oxidase in bovine aortic endothelial cells (BAECs)
Authors: Zhang Quanjiang , Wilson Lloyd , Pettey Dix , Arrant Colton , Kowalski Chris , Abel E Dale , Symons J David
Published: June 2021

In preliminary studies we showed that basal and insulin‐mediated endothelial nitric oxide (NO) synthase phosphorylation (p‐eNOS) and NO production are impaired when BAECs are treated with the saturated fatty acid palmitate (P, 500 uM) vs. vehicle (V) for 3 hours (h). Co‐incubation of BAECs with the ceramide synthesis inhibitor myriocin (M, 10 μM) negated these effects. ROS can lower NO bioavailability. We determined the contribution from ceramide to P‐induced ROS production, and investigated sources of ROS. DCFDA fluorescence was used to assess ROS after BAECs were incubated for 3h with V ± P, M, 10 μM KCN (a mitochondrial ROS inhibitor), or 1 μM NVN (an NADPH oxidase inhibitor). Relative to V, P‐evoked increases (2.8±0.1‐fold, p<0.05, n=48) in ROS production were blunted (15±5%; p<0.05) but were not completely abolished by M, suggesting the existence of ceramide‐dependent and –independent pathways. KCN, NVN, and KCN+NVN lowered (p<0.05) P‐evoked ROS production by 37±4%, 18±7%, and 39±12% (n=8–32), respectively, suggesting that in addition to mitochondrial and NADPH oxidase – dependent sources of ROS, additional sources of palmitate‐induced ROS exist. P‐induced ROS production decreased further when BAECs were co‐incubated with M+NVN (46±4%, p<0.05, n=16) vs. NVN alone, but not when M was co‐incubated with KCN (n=12) or KCN+NVN (n=16). Moreover, P‐evoked increases (p<0.05, n=12) in NADPH oxidase activity were similar in the absence (1.6±0.2‐fold) and presence (2.1±0.2‐fold) of M. Thus, P‐evoked and ceramide‐mediated ROS production in BAECs is not derived from NADPH oxidase but may arise from mitochondria. NIHR15HL091493, ADA7‐08‐RA‐164
S‐Nitrosylation decreases vasodilation via guanylyl cyclase inhibition in mouse aorta
Authors: Choi Hyehun , Webb R. Clinton
Published: June 2021

S‐Nitrosylation (SNO) is a ubiquitous protein modification in redox‐based signaling and forms S‐nitrosothiol with nitric oxide on cysteine residues. We hypothesized that vascular relaxation is decreased by inhibition of guanylyl cyclase (GC) through SNO. Aortic rings from C57BL6 mice were treated with a denitrosylation inhibitor, 1‐chloro‐2,4‐dinitrobenzene (DNCB) for 30 min or S‐nitrosocysteine (CysNO) for 15 min and vascular relaxation was measured in the rings contracted with phenylephrine. In the presence of CysNO, relaxation to acetylcholine (Ach) or sodium nitroprusside (SNP) was decreased and relaxation to GC activator (BAY41‐2272) was increased (Ach: vehicle Emax = 79 ± 3.4 %, CysNO Emax = 54 ± 2.3 %; SNP: vehicle Emax = 98 ± 1.4 %, CysNO Emax = 73 ± 3.8 %; BAY: vehicle pD2 = 6.4 ± 0.08, CysNO pD2 = 7.0 ± 0.06, *p<0.0001, n=6). In DNCB‐treated rings, relaxation to Ach was decreased and relaxation to SNP or BAY41‐2272 was not different from vehicle (Ach: vehicle Emax = 77 ± 1.9 %, CysNO Emax = 53 ± 3.2 %, *p<0.0001, n=7). These data suggest that increased SNO impairs vasodilation via GC inhibition. DNCB is a thioredoxin reductase inhibitor and relaxation to SNP was not different in vehicle and DNCB treatment, suggesting that the thioredoxin system may regulate SNO via the endothelium. Since CysNO acts as exogenous SNO source leading to decrease relaxation to Ach and SNP, augmented SNO by CysNO may work in smooth muscle.
O‐Glcnacylation augments protein kinase C mediated vasoconstriction
Authors: Hardy David , Tostes Rita , Webb Clinton
Published: June 2021

Protein kinase C (PKC) is a key signaling mechanism in vasoconstriction and all PKC isozymes are dynamically modified by O‐linked N‐acetylglucosamine (O‐GlcNAc) in all higher level eukaryotes. We hypothesized that this posttranslational modification of PKC augments vasoconstriction and have reported a 47% increase in contractile response to phenylepherine in rat aorta treated with 100μM O‐(2‐Acetamido‐2‐deoxy‐D‐glucopyranosylidene) amino N‐phenyl carbamate (pugNAc), which selectively increases O‐GlcNAcylation. Co‐immunoprecipitations and western blot analysis demonstrate that PKCα, ‐β, and ‐ε expressed in rat aorta are O‐GlcNAcylated. A selective increase in rat aorta O‐GlcNAc leads to a 139% rise in PKC activity via phosphorylation of PKC specific myristoylated alanine‐rich c kinases (MARCKs). Furthermore, a selective PKC inhibitor (10μM GF109203x) inhibits agonist‐induced contraction in rat aortas treated with pugNAc. In conclusion, O‐GlcNAc augments vasoconstriction by modifying PKC and this post‐translational modification may represent a key regulatory mechanism in the vasculature. Funded by MCG Cardiovascular Discovery Institute.
A role of the novel, non‐AT1, non‐AT2 angiotensin binding site in neuronal cell death
Authors: Rashid Md. Mamunur , Arumugam Thiruma V. , Karamyan Vardan T.
Published: June 2021

The novel, non‐AT1, non‐AT2 angiotensin binding site is a yet unknown protein found in rodent and human brain membranes with pharmacological specificity and tissue distribution different from the type 1 and 2 angiotensin receptors and key enzymes involved in proteolytic processing of angiotensins. Here we report a summary of our recent studies indicating a correlation between the density of the novel binding site in primary neurons and neuronal cell death. 125I‐Sar1, Ile8 angiotensin II binding studies were carried out according to previously established procedures in mouse primary cortical neuronal membranes. Unmasking of the novel angiotensin binding site was carried out by 10 μM p‐chloromercuribenzoic acid (PCMB) in the presence of 10 μM PD123319 and 1 μM ZD7155 (AT1 & AT2 receptor antagonists). 12 in‐vitro‐day‐old primary neurons were challenged in three in vitro models of apoptosis (oxygen‐glucose deprivation for 3 hr, exposure to sodium azide (3 mM) or NMDA (0.5 mM) for 30 min in glucose free EBSS) followed by 24 hr recovery in normal medium. Density of the novel angiotensin binding site in neuronal membranes was estimated by saturation binding assays. The results indicated that the number of novel angiotensin binding sites was significantly elevated in neuronal membranes from all treatment groups, where the viability of neurons was significantly decreased. Supported by TTUHSC SOP start‐up funds to VTK.
Curcumin mitigated ischemic and hyperhomocysteinemic cerebral microvascular mitochondrial mitophagy by decreasing oxidative and inflammatory stresses
Authors: Kapoor Shray , Tyagi Neetu , Qipshidze Natia , Givvimani Srikanth , Kandel Madhavi , Tyagi Suresh C
Published: June 2021

Previous studies have shown that ischemia and hyperhomocysteinemia (HHcy) cause cerebral microvascular permeability; however, it was unclear whether the individuals with genetic HHcy were more prone to brain damage then the individuals with normal levels of homocysteine. Interestingly, curcumin mitigated the inflammation. Therefore we hypothesize that Hcy induced oxidative stress and inflammation after ischemia was ameliorated by Curcumin treatment. To test this hypothesis ischemia was created by inserting a wire in carotid artery in wild type (WT) and cystathionine beta synthase heterozygote knockout (CBS−/+) mice. The ischemic and sham mice were treated with and without curcumin (300 mg/kg) for 4 wks. The levels of mitochondrial mitophagy and oxidative stress were measured by oxidants (p47, p22, gp91phox) and anti‐oxidants (catalase, superoxide dismutase‐2, thioredoxin) by Western blot. The levels COX‐2, ICAM‐1 and PPARgamma were measured as inflammatory markers. The activity of NFkB was measured by EMSA. The results suggested that there was increase in p47, p22 in the ischemic and HHcy mice. Curcumin treatment mitigated mitochondrial mitophagy, in part, by increasing in NFkB activation. We concluded that Hcy caused oxidative stress and inflammation after ischemia. The treatment of curcumin ameliorated the mitophagy, oxidative stress and inflammation in both the ischemia and HHcy mice.
Dihydrotestosterone attenuates HIF‐1alpha and COX‐2 in cerebral arteries following hypoxia.
Authors: Osterlund Kristen Leanne , Handa Robert J , Gonzales Rayna J
Published: June 2021

Vascular inflammation is under the control of several transcription factors, one of which is hypoxia inducible factor‐1 alpha (HIF‐1α). HIF‐1α activation can lead to the production of inflammatory mediators such as cyclooxygenase‐2 (COX‐2). This study determined the influence of dihydrotestosterone (DHT) on HIF‐1α and COX‐2 following hypoxia. COX‐2 and HIF‐1α protein levels were assessed in cerebral arteries isolated from castrated rats treated with DHT (3 wk) in vivo and exposed to normoxia or hypoxia (1%; 6h) ex vivo and in primary human brain vascular smooth muscle cells pretreated with vehicle or DHT (100nM; 18h) and exposed to hypoxia and glucose deprivation (1% O2; 0 or 6h). Under normoxic conditions, DHT increased COX‐2 levels but had no effect on HIF‐1α protein levels and HIF‐1α DNA‐binding was below the level of detection. Following hypoxia or hypoxia and glucose deprivation, HIF‐1α and COX‐2 levels were increased; this response was blunted in the presence of DHT. In addition, increases in HIF‐1α DNA‐binding following hypoxia (1%; 1h) were reversed by DHT. In conclusion, under hypoxic conditions, the blunted response of COX‐2 by DHT may involve HIF‐1α activation. In contrast, the effect of DHT to increase COX‐2 under normoxic conditions may be independent of the HIF‐1α pathway.

AHA SDG RG, AHA Predoctoral Fellowship KO
Antenatal Steroid Exposure is Associated with Reduced Urinary ACE2 Activity in Adolescents
Authors: Gwathmey TanYa M. , Shaltout Hossam A. , Nixon Patricia A. , Rose James C. , Washburn Lisa K. , Chappell Mark C.
Published: June 2021

The therapeutic use of antenatal administration of glucocorticoids is now common for women at risk of pre‐term delivery. However, both human and animal studies have reported that offspring of the treated mother exhibit increased mean arterial pressure (MAP) by adulthood. To evaluate a potential role for the renal renin angiotensin system (RAS) in humans, we assessed angiotensin (Ang) metabolism in urine collected from adolescents (14 years old) exposed antenatally to betamethasone (BMX; n=18) compared to that of non‐exposed individuals (CON; n=25). ACE activity, determined by the hydrolysis of radiolabeled 125I‐Ang I to 125I‐Ang II was not significantly different between groups (BMX: 0.71±0.10 vs. CON: 0.93±0.12 fmol/min/g creatinine). However, conversion of 125I‐Ang II to 125I‐Ang (1–7) by ACE2 was reduced by antenatal BMX exposure (BMX: 0.66±0.16 vs. CON: 1.09±0.17 fmol/min/g creatinine; P=0.04). MAP did not differ between groups at this age (BMX: 76±1 vs. CON: 75±1 mmHg). These data reveal that adolescents exposed to antenatal BMX treatment exhibit reduced urinary ACE2 activity in the absence of alterations in MAP. Moreover, the present study supports our findings in a sheep BMX model of fetal programming in which tubular and urinary ACE2 activity was reduced. We conclude that alterations in urinary (renal) ACE2 during adolescence may serve as a biomarker for the development of hypertension in adulthood.
Renin‐Angiotensin‐Aldosterone System During Extracorporeal Membrane Oxygenation (ECMO) in a Pig Model of Septic Shock
Authors: Mu Thornton Samuel , Batts Sherreen G. , Sato Aileen , Hernandez Claudia , Ichimura Wayne , Lentz‐Kapua Sarah L , Uyehara Catherine F.T.
Published: June 2021

ECMO is used to provide cardiopulmonary support to critically ill patients, but the ability of the renin‐angiotensin‐aldosterone system to function appropriately during ECMO remains unclear. In this study, plasma renin activity (PRA) and aldosterone (ALDO) were compared at baseline and before and after stabilization on veno‐arterial ECMO for 2 hours in anesthetized piglets either during control (n=10) or endotoxin‐induced septic shock (n=9) states. In control piglets, ECMO was able to maintain mean arterial pressure (MAP) at baseline levels and PRA and ALDO remained unchanged. In contrast, endotoxin induced a 46% decrease (p<0.01) in MAP, and while ECMO could maintain flow at this decreased MAP, it did not return MAP to baseline. PRA appropriately increased in response to shock, but ultimately dropped by 54% (p<0.01) in early ECMO. ALDO increased in response to shock, and remained constant during ECMO. There was a strong correlation between PRA and ALDO at baseline (r=0.90, p<0.01) and on ECMO in control piglets (r= 0.70, p<0.01), but septic shock disrupted that relationship. Plasma sodium concentrations increased (p<0.01) from 137 ± 3.3 to 145 ± 0.7 mEq/L in shock, and with sustained ALDO, remained at this elevated level throughout ECMO. Results suggest that ALDO sodium regulating ability remains intact during ECMO but PRA is attenuated during ECMO in the endotoxic shock state.
An essential role of cortactin in regulation of ENaC
Authors: Ilatovskaya Daria , Vinnakota Kalyan C , Pavlov Tengis S , Staruschenko Alexander
Published: June 2021

Epithelial Na+ Channel (ENaC) is critical for maintenance of sodium balance. Cortactin is a key signaling protein for many cellular processes, including direct interaction with F‐actin. In this study we have addressed the role of cortactin in mediating regulation of ENaC. As assessed by Western blotting, cortactin is highly expressed in mpkCCDc14, M‐1 and MDCK cells. Immunohistochemistry analysis showed localization of cortactin in the cortical collecting duct in Sprague‐Dawley rat kidneys. Co‐immunoprecipitation analysis revealed that cortactin directly interacts with ENaC subunits when co‐expressed in CHO cells. We have supported these data with fluorescent microscopy co‐localization approach and fluorescence correlation spectroscopy analysis. Coexpression of cortactin with ENaC decreased channel's activity as measured by patch‐clamp. Surprisingly, cortactin's dynamin‐binding Src homology 3 domain is not required for ENaC regulation. Similarly cortactin mutants with deleted phosphorylation domain, or lacking ability to bind F‐actin decrease ENaC current. However cortactin mutant unable to bind Arp2/3 complex does not influence ENaC activity. Thus, we conclude that cortactin decreases ENaC activity via Arp2/3 complex.

Supported by AHA and ASN.
Different Types of Vesicle Trafficking of NBCe1 (SLC4A4)
Authors: Grichtchenko Irina I. , Perry Clint J. , Bellis Aaron Alen
Published: June 2021

We previously reported that basolateral NH2‐terminal NBCe1‐A and NBCe1‐B variants of NBCe1 (encoded by SLC4A4 gene) is regulated by internalization/endocytosis induced by signaling of ANGII via AT1R or CCh via M3R receptors (AJP Renal 2006, 2007, AJP Cell, 2008, 2009). Here we report that internalized endogenous NBCe1 and recombinant NBCe1 variants tagged to Green Fluorescent Protein (NBCe1‐GFP) are localized to same intracellular trafficking vesicles as Texas Red‐conjugated ligand of transferrin receptor (Tfn‐TR). We also found that inhibition of dynamin‐dependent endocytosis with dynasore prevents loss of NBCe1 variants from the cell surface. These data suggest that internalization of NBCe1 variants is clathrin‐ and dynamin‐dependent and that cytoskeleton plays an important role in this fast mode of endocytosis. Next, we found that some of endosomal formations that contain internalized NBCe1 variants are marked with anti‐Rab11 (marker of recycling endosomes) and some with anti‐LAMP1 (marker of late endosomes). Therefore, we show that the intracellular trafficking process of NBCe1 variants do not stop in early endosomes (co‐localization with EEA1), but continue on to be recycled via fast or slow mode of vesicle trafficking. These new observations taken together with our previous reports suggest that NBCe1 variants undergo near‐membrane cycling via several vesicle trafficking mechanisms.
Defining roles for P2Y2 and P2Y4 receptors on blood pressure and urinary excretion
Authors: Rieg Timo , Schroth Jana , Insel Paul A , Broyer José , Vallon Volker
Published: June 2021

Mice lacking P2Y2 receptors (P2Y2−/−) have an increased blood pressure (BP) and impaired renal excretion of salt and water. To define the in vivo effect of P2Y2 receptor activation on BP and urinary excretion, we compared the effects of the P2Y2/4 receptor agonist, INS45973, in wild‐type (WT) and P2Y2−/− mice (n=8/genotype) under anesthesia. Acute application of INS45973 (0.1, 0.3, 1, or 3 mg/kg bw i.v. in 1 ml/kg bw over 45 sec) dose‐dependently and rapidly (within 15 s of starting infusion) decreased BP in WT (max response −35±2 mmHg) for 2–3 min. In P2Y2−/−, INS45973 increased BP (max response +18±1 mmHg). Continuous infusion (25, 75, 250, or 750 μg/min/kg bw i.v. for 15 min with quantitative urine collection over the last 10 min) did not affect BP in WT or P2Y2−/−. In WT, INS45973 dose‐dependently increased urinary excretion of Na+ (max response +46±15%); the excretion of fluid and K+ were not different compared to vehicle. In P2Y2−/−, INS45973 reduced urinary excretion of Na+, K+ and fluid (max responses of −45±15%, −50±6%, and −37±7%, respectively). The acute BP responses indicate P2Y2 receptor‐mediated vasodilation and P2Y4 receptor‐mediated vasoconstriction. The renal responses are consistent with an inhibitory influence of P2Y2 receptor activation on renal Na+ reabsorption and a pro‐reabsorptive effect of P2Y4 receptor activation. P2Y2 and P2Y4 receptors thus appear to functionally antagonize one another.
ENaC Expression Alters a Recycling Vesicle Pool Responsible for its Regulation
Authors: Butterworth Michael , Edinger Robert , Bertrand Carol , Frizzell Raymond , Johnson John
Published: June 2021

The epithelial sodium channel (ENaC) is the rate limiting step in sodium reabsorption across tight epithelia. In kidney cortical collecting duct (CCD) cells cAMP stimulation increases Na+ transport by trafficking ENaC from an intracellular vesicular pool to the apical surface. By reducing the activity of the serum and glucocorticoid kinase (SGK) in a cultured CCD cell line we demonstrate that both ENaC expression and the size of the vesicle‐trafficking pool responsible for ENaC trafficking is reduced. Selective stimulation of SGK with aldosterone or dexamethazone increases both the basal and cAMP‐stimulated ENaC short‐circuit currents (ISC) and the size of the ENaC‐containing vesicle pool enlarges. Fusion of ENaC‐containing vesicles with the apical membrane is measured as a change in transepithelial capacitance (CT). The cAMP‐induced ΔCT in unsupplemented, low ENaC expressing cells was 0.11±0.008 μF/cm2 which increased to 0.15±0.01 μF/cm2 in aldosterone supplemented cells. To determine if the size of the vesicle pool depends on ENaC alone or another downstream component of the SGK signaling pathway, αENaC was selectively knocked down in CCD cells by siRNA. ENaC‐dependent ISC was significantly reduced from 12.9±3.0μA/cm2 in control cells to 1.95±0.36μA/cm2 in the αENaC knockdown cells. There was also a significant reduction in the cAMP‐stimulated ΔISC and ΔCT suggesting that ENaC alone determined the size of the exocytosed vesicle pool. To verify that ENaC modulates this exocytosed vesicle pool, polarized Fischer Rat Thyroid (FRT) cells which do not endogenously express ENaC were transfected with α,β and γ‐ENaC constructs. The CT response to cAMP was significantly greater in cells transfected with ENaC. Together these results indicate the presence of a recycling vesicle pool which is regulated by ENaC expression.
Vesicle associated membrane protein (VAMP) 3 but not VAMP2 is involved in NKCC2 apical targeting and processing in thick ascending limbs (TALs)
Authors: Caceres Paulo S , Mendez Mariela , Ortiz Pablo A
Published: June 2021

NKCC2 undergoes apical targeting to the cell surface in TALs in the absence of hormonal stimulation. VAMP2 and VAMP3 colocalize with NKCC2 in the apical membrane and subapical space of TALs and may differentially regulate protein processing, sorting and apical targeting of NKCC2. We hypothesized that both VAMP2 and VAMP3 regulate constitutive NKCC2 surface expression. We measured the effect of a) siRNAs against VAMP2 and VAMP3 and b) tetanus toxin; on surface and total NKCC2 expression by biotinylation of rat TAL suspensions and Western blot. VAMP2 siRNA decreased VAMP2 (85% decrease) without affecting VAMP3 and VAMP3 siRNA decreased VAMP3 (95% decrease) without affecting VAMP2 in control experiments. VAMP2 siRNA did not affect surface NKCC2 (81 ± 7 % of control) or total NKCC2 expression (93 ± 9 % of control). In contrast, VAMP3 siRNA decreased surface NKCC2 by 53 ± 13% (p<0.05) and total NKCC2 expression by 59 ± 8 % (p<0.01), suggesting that VAMP3 mediates both processing and apical targeting. We next used a low concentration of tetanus toxin, which cleaves and inactivates VAMP2 and VAMP3, to block apical targeting, but not processing or sorting. Fifty min treatment with 30 nM tetanus toxin decreased surface NKCC2 by 33 ± 5 % (p<0.05) without affecting total NKCC2 expression. Our data suggest that VAMP3 is involved in both NKCC2 processing and apical targeting in TALs whereas VAMP2 is not likely involved in these processes.

NIH
Mutations in renal drug efflux transporter hOATv1 cause hyperuricemia
Authors: Jutabha Promsuk , Anzai Naohiko , Kitamura Kenichiro , Taniguchi Atsuo , Kaneko Shuji , Yamada Hideomi , Urano Wako , Seki George , Endou Hitoshi , Sakurai Hiroyuki
Published: June 2021

Hyperuricemia, traditionally linked to gout, has recently been implicated in cardiovascular diseases and hypertension. One of the major determinants of plasma urate concentration is renal excretion that is dependent on the balance between reabsorption and secretion. Previously we identified that renal urate absorption is mainly performed by apical urate/anion exchanger, URAT1/SLC22A12, and basolateral voltage‐driven urate efflux transporter, URATv1/SLC2A9 in tandem in proximal tubular cells. In contrast, the molecular mechanism of renal urate secretion remains elusive. Here, we report that newly characterized human renal voltage‐driven drug efflux transporter, hOATv1 also functions as a long‐hypothesized urate exit pathway at the apical side of renal proximal tubules that determines the direction of net urate flux. Loop diuretics such as furosemide and bumetanide substantially interacted with hOATv1, providing the first molecular evidence for loop diuretics‐induced hyperuricemia. The in vivo role of hOATv1 is supported by the fact that missense mutations in this transporter found in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Our results demonstrate that hOATv1 is component of renal urate secretory pathway involved in the formation of both in primary and secondary hyperuricemia.
Angiotensin II Stimulates ENaC and suppresses the inhibitory effect of 11,12‐EET on ENaC in the cortical collecting duct (CCD)
Authors: Sun Peng , Lin Daohong , Yue Peng , Wang Wenhui
Published: June 2021

We used the patch‐clamp technique to study the effect of angiotensin II (Ang II) on ENaC in principal cell (PC) of the CCD. The amiloride‐sensitive Na currents in PC measured with perforated patch were 480¡A64 pA at −100 mV in rats on a high K (10%) diet. Application of 100 nM Ang II increased Na current by 260% to 1247¡A289 pA. However, treatment of the CCD with losartan abolished Ang II‐induced increase in Na current, suggesting that the stimulatory effect of Ang II on ENaC was the result of activating Ang II type I receptor (AT1R) which is expressed in the CCD. The stimulation of Ang II on ENaC was also observed by single channel recording: Ang II increased the ENaC activity defined by NPo from 0 to 0.4¡A0.08 within 5 min. We confirmed that addition of 100 nM 11,12‐EET (a major product of CYP epoxygenase‐dependent arachidonic acid metabolism) inhibited ENaC in the CCD in rats on a high K diet. However, the supression of EET on ENaC was significantly attenuated by application of Ang II which increased EC 50 value of 11,12‐EET‐induced inhibition from 50 to 100 nM. We conclude that acutely application of Ang II stimulates ENaC activity via AT1R‐dependent pathway and diminishes the inhibition of 11,12‐EET on ENaC.