bluuebunny/crossref_metadata_2025 huggingface dataset:
Antidepressant‐like effect of AMPA and ketamine combination in male Wistar ‐Kyoto rats
Authors: Akinfiresoye Luli , Lovinger D. M. , Tizabi Y.
Published: June 2021

Studies have shown that therapeutics that act on the glutamatergic system could provide a novel mechanistic approach for the treatment of depression. We previously reported that ketamine, an NMDA antagonist, has a rapid and lasting antidepressant effect both acutely and chronically on depressive‐like behavior in Wistar‐Kyoto (WKY) rats (Soc Neurosci Abst 2008). Other studies had suggested that the antidepressant effect elicited by ketamine could be mediated through stimulation of AMPA receptors, we also sought to confirm this hypothesis in our animal model of depression. We discovered that AMPA by itself also has an antidepressant effects in WKY rats, both acutely and chronically (Biol Psych Abst 2010). In this study we examined the hypothesis that the combination of AMPA and ketamine will result in either a synergistic or additive antidepressant effect when compared to the individual treatments of AMPA or ketamine alone. Adult male WKY rats were administered the combination of AMPA (0.25 mg/kg, i.p.) and ketamine (0.25 mg/kg, i.p.) for 10 days and behavior studies were done approximately 22 hr after the last injection. Controls received saline. A 5 minute forced swim test (FST), a measure of helplessness, was used to determine the drug‐induced antidepressant effects. The combination of AMPA and ketamine resulted in a significant reduction of FST immobility compared to individual treatment of either AMPA or ketamine alone. In view of the fact that an increase in AMPA/NMDA receptor function and number may be mediating the antidepressant effects of ketamine, these results suggest that a combination of AMPA and ketamine could provide an effective therapeutic treatment for depression.

Supported by NIH/NIGMS (2 SO6 GM08016‐39)
Benzodiazepine actions: Teasing apart drug binding from drug efficacy
Authors: Morlock Elaine V. , Hanson Susan , Czajkowski Cynthia
Published: June 2021

Benzodiazepines (BZDs) are clinically important drugs that exert their CNS actions by binding to the GABAA receptor (GABAAR) and allosterically modulating GABA‐activated chloride currents (IGABA). Their actions depend on residues in the BZD binding site that mediate their high‐affinity binding as well as residues that mediate local movements in the site important for coupling BZD binding to modulation of IGABA (efficacy). To identify and distinguish residues involved in each of these functions, we made 22 mutations in and surrounding the BZD binding pocket. To assess BZD binding, mutant α1β2γ2 GABAARs were expressed in HEK293T cells and radioligand binding assays used to measure BZD affinity. To identify residues that contribute to drug efficacy, mutant receptors were expressed in Xenopus laevis oocytes, characterized using two‐electrode voltage clamp and BZD maximal potentiation of IGABA was measured. We identified six residues whose mutation altered BZD efficacy without altering BZD binding affinity, three residues whose mutation altered binding but had no effect on efficacy, and four residues whose mutation affected both binding and efficacy. These data advance our understanding of allosteric modulation and will aid in future rational drug design.
Acute Modulation of Voltage‐Gated Ca2+ Channels in Retinal Ganglion Cells by Gabapentin
Authors: Farrell Spring R , de Sevilla Mueller Luis Perez , Sargoy Allison , Brecha Nicholas , Barnes Steven
Published: June 2021

The α2δ1 auxiliary subunits of voltage‐gated Ca2+ channels (VGCCs) are important modulators of VGCC function. Gabapentin interacts with α2δ1 subunits. Thus, it is possible that gabapentin may prevent the deleterious effects of elevated Ca2+ levels in retinal ganglion cells (RGCs) in trauma and disease. The aim of this study was to establish the expression of α2δ1 VGCC auxiliary subunits in the retina and to determine the effects of gabapentin on VGCCs in RGCs. Immunohistochemistry was performed on rat retinal sections to determine the localization of α2δ1. Patch clamp was used to record Ca2+ currents (ICa) in isolated RGCs. ICa was measured in the absence and presence of gabapentin. Retinal sections exhibited α2δ1‐like immunoreactivity in RGC cell bodies and their primary dendrites. In isolated RGCs, acute administration of gabapentin reduced ICa compared to control. Pretreatment of RGCs with gabapentin reduced ICa compared to non‐pretreated cells. Also, acute administration of gabapentin to RGCs that had been pretreated further reduced ICa. Thus, modulation of α2δ1 VGCC subunits may prove to be an important target in the neuroprotection of damaged RGCs. Ongoing work is aimed to determine the mechanism of acute modulation of VGCCs by gabapentin.
Enhanced relaxant effect of Sodium Hydrogen Sulfide (NaHS) in Experimental Colitis and its action on KATP Channels via S‐sulfhydration
Authors: Kang Minho , Gade Aravind , Akbarali Hamid I
Published: June 2021

Hydrogen sulfide (H2S) has recently been recognized as a gaseous signaling molecule that affects smooth muscle function. The aim of this study was to determine the effect of inflammation on the responses to H2S in smooth muscle of mouse colon. Colonic inflammation was induced by intracolonic administration of trinitrobenzene sulfonic acid (TNBS) in BALB/C mice. Isometric force was measured in the organ bath. In carbachol‐precontracted tissues, levcromakalim (LEV), a KATP channel opener, induced dose‐dependent relaxations (IC50 1036 nM, n=5). The dose‐response was shifted to the left in inflamed colon (IC50 458 nM, n=6). Similarly, relaxations to NaHS were also shifted to the left in the colitis model (control IC50 196 μM, n=6: inflamed IC50 151 μM, n=3) and maximal amplitude of relaxation was enhanced by 40%. Pretreatment with glybenclamide (100 uM) abolished LEV relaxations and reduced NaHS relaxations by 46 ± 8.2% (n=3). A Biotin switch assay was performed to determine s‐sulfyhdyration of SUR2B subunit of KATP. NaHS significantly enhanced sulfhyrdration of SUR2B in CHO transfected cells and in native colonic tissues. These data suggest that enhanced hydrogen sulfide during colitis, regulates contractile activity in the smooth muscle of mouse colon through S‐sulfhydration of the KATP channel. Supported by NIH DK046367.
C terminal block of the Pannexin 1 channel pore and its relief by proteolytic cleavage
Authors: Sandilos Joanna K , Chiu Yu-Hsin , Chekeni Faraaz B , Armstrong Allison J , Walk Scott F , Ravichandran Kodi S , Bayliss Douglas A
Published: June 2021

Pannexin‐1 (PANX1) channels release ATP, a “find‐me” signal that recruits macrophages to apoptosing cells. Cleavage of the PANX1 C terminus by caspases represents a new mechanism of ion channel opening, but how the C terminus regulates channel activity is unclear. We provide evidence suggesting that the C terminal residues of PANX1 block the pore and removing this block activates the channel. Silent channels could be activated directly by C terminal cleavage, in the absence of additional apoptotic factors. We identified a region immediately downstream of the caspase site required for inhibition of PANX1. Consistent with the C terminus acting as a pore blocker, we found that constitutively active channels could be inhibited, in trans, by the isolated C terminus. Furthermore, using a cysteine crosslinking approach, we show that relief of inhibition following cleavage requires dissociation of the C terminus from the channel pore. Together, these data suggest a ball & chain mechanism of PANX1 activation, wherein the C terminus blocks the full length channel and a remarkably well placed caspase cleavage site allows effective removal of inhibitory C terminal residues to facilitate channel opening.
Effect of arsenic and arsenic metabolites on LTCC and BKCa expression and activities in vascular smooth muscle
Authors: McPherson Kevin , Pace Clare , Khalili Roxana , Angermann Jeff Erik
Published: June 2021

Background

Arsenic (As) contamination of ground‐ and well‐water is common worldwide. Chronic As exposure can produce toxic effects including atherosclerosis and hypertension, yet mechanisms by which both inorganic As and As metabolites induce these effects are not well elucidated. Both arsenite (iAs3+) and its metabolite monomethylarsonous acid (MMAs3+) are believed to affect the activity of critical Vascular Smooth Muscle (VSM) components including the ‘L‐type’ calcium channel (LTCC), a key regulator of VSM tone, as well as the large‐conductance calcium‐activated potassium channel (BKCa), a known modulator of cell depolarization implicated in the development of hypertension and restenosis disease.

Objectives and Methods

This study examines effects of iAs3+ and MMAs3+ on expression and related activities of LTCC and BKCa in acutely isolated and primary / tissue cultured rat thoracic aorta, as well as the A7r5 rat thoracic aorta VSM cell line, using whole‐cell patch clamp, vascular contractility, RT‐QPCR, and light microscopy.

Results

Initial results indicate alterations in VSM cell morphology, viability, LTCC activity, and responsiveness to PE‐induced vasoconstriction on acute and subchronic exposure to both iAs3+ and MMA3+.

Conclusions

Both iAs3+ and MMA3+ affect activities of key effectors governing the maintenance of VSM tone. This work was supported by the Nevada INBRE and McNair Programs.
Characterization of PPARγ ligand MCC‐555 in AOM‐induced colorectal tumorigenesis
Authors: Imchen Temjenmongla , Min Kyung-Won , Baek Seung Joon
Published: June 2021

PPARγ agonists affect cell proliferation, differentiation, and apoptosis in a PPARγ dependent and/or‐independent manner, and thereby represent a potentially important family of therapeutic compounds for cancer treatment. Dual ligands for PPARα and PPARγ, such as MCC‐555, have been developed to improve treatment of metabolic syndrome, including hyperglycemia and hyperlipidemia. Interestingly, these dual ligands also possess anti‐proliferative activities against a variety of cancer cell lines with greater potency than conventional PPARγ specific ligands. In this study, colon cancer chemopreventive properties of MCC‐555, were evaluated using azoxymethane (AOM)‐induced colonic aberrant crypt foci (ACF) in female A/J mice. 36 female mice were randomly assigned to 5 groups: (1) negative control treated with carboxymethylcellulose (vehicle); (2) positive control treated with carboxymethylcellulose plus AOM; (3) treated with 30 mg/kg MCC‐555 plus AOM; (4) treated with 60 mg/kg MCC‐555 plus AOM; (5) treated with 60 mg/kg MCC‐555. At 6 weeks of age, all animals were gavaged with carboxymethylcellulose or MCC‐555 for 5 weeks starting 1 week before initiation with AOM injection. AOM was injected i.p. once a week for 4 weeks at 10 mg/kg for groups 2, 3 and 4. Animals will be dissected, and ACF will be examined. The results will be discussed at the meeting.

This work was supported by NIH grant‐R01CA108975 and University of Tennessee Center of Excellence in Livestock Diseases and Human Health grant. Temjenmongla Imchen is a recipient of National Overseas Scholarship Award [No. 11016/07/2010‐Education] from Ministry of Tribal Affairs, New Delhi, India.
Decreased Renal and Urinary Neprilysin Protein Expression in db/db Mice
Authors: Chodavarapu Harshita , Bradshaw Melissa , Salem Esam , Elased Khalid
Published: June 2021

The renin angiotensin system (RAS) plays a pivotal role in the pathogenesis of diabetic nephropathy. Neutral endopeptidase or neprilysin (NEP) belongs to the M13 family of zinc‐containing metallopeptidases which converts Ang I to Ang (1–7). Ang (1–7) is a potential target in renal pathology because of its opposing effects on Ang II induced actions. The possible involvement of NEP in diabetic renoprotection has not been investigated; therefore, the aim of this study is to examine the role of neprilysin in the pathogenesis of diabetic nephropathy in db/db mice. db/db mouse model for type 2 diabetes was used to study the effect of diabetes on renal and urinary NEP. These mice exhibit hyperglycemia, hypertension, hyperinsulinemia, obesity and microalbuminuria. Urine was collected over 24 hours from db/db and lean control mice to measure the urinary albumin, creatinine and total protein. Our results showed that db/db mice were hyperglycemic and demonstrated an early onset of microalbuminuria. Western blot analysis revealed decreased renal and urinary NEP protein expression in db/db mice compared to lean control mice (p<0.05). Immunohistochemical staining of kidney sections revealed a decrease in NEP protein expression in the db/db mice. In conclusion, downregulation of NEP could play an important role in the development of diabetic nephropathy in db/db mice.

Supported by the American Heart Association.
Effect of gum Arabic on oxidative stress and inflammation in adenine–induced chronic renal failure in rats
Authors: Ali Badreldin H. , Al-Husseni Isehaq , Beegam Sumyia , Al Shukali Ahmed A. , Nemmar Abderrahim , Schupp Nicole
Published: June 2021

Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in surgically‐induced chronic renal failure (CRF) in rats. Evidence for the involvement of these processes in adenine‐induced CRF in rats is lacking. The aim of this work was to study the role of inflammation and oxidative stress in adenine‐induced CRF, and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in the feed (0.75%, w/w), GA in drinking water (15%,w/v) and adenine + GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration and urinary NAG activity). In addition, the concentrations of several pro‐inflammatory cytokines and indices of oxidative stress, renal apoptosis, superoxide formation and DNA double strand break frequency detected with immunohistochemistry for γ‐H2AX were also conducted in the kidney tissue, plasma and urine. Adenine (0.75% in the feed for 4 weeks) caused significant increases (P < 0.001) in the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance (P < 0.01) and induced significant increases in the concentration of the measured inflammatory mediators. Treatment with GA significantly ameliorated these actions, suggesting that the mechanism of the reported salutary effect of GA in adenine‐induced CRF is associated with mitigation of the adenine‐induced inflammation and generation of free radicals.
Hepatic PXR represses UGT1A1 gene expression during neonatal development
Authors: Chen Shujuan , Tukey Robert H.
Published: June 2021

Bilirubin glucuronidation is catalyzed solely by UDP‐glucuronosyltransferase 1A1. The delayed development of UGT1A1 expression in neonates leads to hyperbilirubinemia. Under conditions of extreme hyperbilirubinemia, bilirubin induced neurological dysfunction (BIND) can result in irreversible brain damage. To understand those events that regulate UGT1A1 gene expression, our laboratory has reproduced neonatal hyperbilirubinemia in humanized UGT1 (hUGT1) mice. The UGT1A1 gene is regulated by both the pregnane X receptor (PXR) and constitutive androstane receptor (CAR). In hUGT1/Car−/− mice, neonates become susceptible to the development of BIND, without alterations in hepatic UGT1A1 expression. Thus, CAR plays an important role in bilirubin access to the CNS. However, hUGT1/Pxr−/− mice are associated with a reduction in bilirubin and upregulation of hepatic UGT1A1 gene expression, suggesting that PXR represses UGT1A1 gene expression during neonatal development. This was confirmed by CHIP analysis, which demonstrated intense binding of PXR to the UGT1A1 gene. In primary hepatocytes isolated from neonatal hUGT1 mice transfected with PXR specific siRNA, de‐repression of UGT1A1 expression occurred following the arrest of PXR synthesis. Thus, PXR represses UGT1A1 gene expression in neonatal mice leading to the onset of hyperbilirubinemia. (Supported by grant GM086713)
Ventricular epicardial repolarization pattern in diabetic rabbits
Authors: Azarov Jan E. , Ovechkin Alexey O. , Vaykshnorayte Marina A , Sedova Ksenia A. , Shmakov Dmitry N.
Published: June 2021

The purpose of the study was to find out the spatial pattern of ventricular repolarization in diabetes mellitus (DM) in heart in situ and to estimate the contribution of sympathetic tone to its development.

DM was induced in rabbits with alloxan (100 mg/kg). At 4–5 week follow‐up, the ventricular electrical potentials were mapped with the 64‐electrode epicardial sock array in zoletil‐anesthetized (10 mg/kg) 13 diabetic and 8 healthy animals. Activation‐recovery intervals (ARIs) serving as repolarization duration indices were measured in each lead prior to and after the injection of esmolol (0.5 mg/kg).

The left ventricular (LV) apical ARIs were shorter than the basal ARIs in controls (P<0.046), whereas the converse was found in DM (P<0.022). Additionally, an interventricular ARI gradient was found in DM group with the right ventricular (RV) ARIs being longer than the LV ARIs (P<0.022). Esmolol abrogated all the repolarization gradients within the ventricles in healthy and DM rabbits by the preferential prolongation of the initially shorter repolarization durations.

Thus, DM caused the remodeling of ventricular repolarization which at least in part could be ascribed to the sympathetic dysfunction.
Effect of body position and hypoxia on the recruitment of the patent foramen ovale
Authors: Moses Kayla , Bates Melissa , Beshish Arij , Pegelow David , Eldridge Marlowe
Published: June 2021

The patent foramen ovale (PFO) is a recruitable intracardiac shunt between the right and left atrium. We aimed to determine if body position and oxygen tension affect blood flow through the PFO in healthy adults. We hypothesized that hypoxia and body positions that promote right atrial filling would independently recruit the PFO. Subjects with (n=3) a PFO performed 11 trials, combining four different O2 levels (100, 21, 15, or 10%) and three positions (upright, supine, and 45° head down), with the exception of 10% O2 while 45° head down. After five minutes in each position, breathing the prescribed oxygen tension, saline bubbles were injected into an antecubital vein and a four‐chamber echocardiogram was obtained. Bubble density passing through the PFO was quantified using a 0–5 scale. We found that changes in oxygen tension and body position had little independent effect on PFO recruitment (2±1). However, PFO recruitment increased when breathing 15 and 10% O2 in the supine position (3±1). This suggests that the combination of hypoxia and position may increase right atrial pressure, increasing right‐to‐left shunting across a PFO. We expected that elevated right atrial pressure would cause PFO recruitment to be greatest in the head down position while breathing hypoxia, but were surprised to find no effect (1±1). We plan to continue these studies and further investigate these unanticipated findings.
Increased angiogenesis as a mechanism for the preserved cardiac function in rats with chronic pressure overload
Authors: Gao Shumin , Park Misun , Tian Yimin , Yan Lin , Lin Lin , Zhao Xin , Gelpi Ricardo , Vatner Dorothy E , Vatner Stephen F
Published: June 2021

Reduced coronary reserve and left ventricular (LV) function are hallmarks of chronic pressure overload. In rats with 4–8 wks of transverse aortic constriction (TAC), the LV/aortic pressure gradient (145±7mmHg), was greater than that observed in most other models of TAC, but LV systolic wall stress was not significantly increased and LV ejection fraction, measured by echocardiography, was not decreased, but actually was enhanced, p<0.05, compared with sham rats at 8 wks (79±3% vs. 73±1%). Coronary reserve, the increase in coronary flow velocity with adenosine measured echocardiographically, which is depressed in almost all models of LV hypertrophy, was preserved in TAC rats vs. sham. There was a significant increase, p<0.05, in vascular endothelial growth factor (VEGF), measured by western blotting (11.9 vs. sham 3.3) and there were increased, p<0.05, Ki67 positive endothelial cells in TAC rats (22±3 Ki67 positive/mm2) vs. sham (12±2 Ki67 positive/mm2), indicative of angiogenesis. Capillary density, quantified microscopically with isolectin staining, was also increased in TAC (480 units/mm2) vs. sham (397 units/mm2). Thus, this unusual model of chronic pressure overload LV hypertrophy in the rat demonstrated enhanced LV function and preserved coronary reserve, which in turn could be attributed to the increased angiogenesis.
Estrogen induces cardiomyocyte contractility via an MLCK/MRLC‐dependent mechanism
Authors: Kararigas Georgios , Bito Virginie , Sipido Karin R , Regitz-Zagrosek Vera
Published: June 2021

Activation of myosin regulatory light chain (MRLC) by MRLC kinase (MLCK) is crucial for actin/myosin interactions and modulation of contraction. The steroid hormone 17β‐estradiol (E2) is mainly considered cardioprotective. However, little is known about E2 effects on contractile and regulatory proteins and cardiomyocyte (CM) contraction. We hypothesized that E2 induces the phosphorylation of MRLC via MLCK increasing CM contractility. Genome‐wide expression profiling of female human cardiac tissues treated with E2 showed a modest but significant increase in the expression of MLCK encoded by MYLK3 compared to controls. Next, we treated middle‐aged female mice with 0.2 mg/kg E2 and isolated CMs enzymatically using the Langendorff system. Western blotting revealed higher phosphorylation of MRLC with E2. Unloaded cell shortening measurements showed that E2 increased contractility. Subsequently, we employed the HL‐1 CM line to inhibit MLCK pharmacologically with ML‐7. As expected, E2 increased the phosphorylation of MRLC in HL‐1 CMs. However, ML‐7 inhibited the E2 effect on MRLC phosphorylation. Our work is the first to show increasing effects of E2 on cell contraction via an MLCK/MRLC‐dependent mechanism.
Control of the steelhead trout (Oncorhynchus mykiss) coronary microcirculation: Temperature effects and role of the endothelium
Authors: Costa Isabel A. S. F. , Hein Travis W. , Secombes Christopher J. , Gamperl A. Kurt
Published: June 2021

The effect of temperature on the local control of microvessel resistance in fish is poorly understood, plus the influence of prostaglandins (PG) and nitric oxide (NO) on vascular smooth muscle in these taxa is controversial. Thus, we measured the dose‐dependent responses of isolated coronary microvessels (< 150μm diameter) from 10, 5 and 1°C acclimated trout to several vasoactive compounds. While vessel sensitivity to most compounds was reduced at 1°C, cold exposure altered the response of vessels to low acetylcholine concentrations and increased the sensitivity of vessels to endothelin‐1. We also found that the dosedependent dilation caused by the endothelium dependent Ca2+ ionophore A23187 was abolished by indomethacin (Indo) and L‐NIO, cyclooxygenase and NO synthase inhibitors, respectively. This suggests a possible role for both PG and NO pathways in endothelial derived regulation of vascular resistance in this tissue. Finally, interleukin (IL)‐1β is linked with hypotension and cardiovascular collapse in mammals during heatstroke, and may have similar effects in fish. Isolated trout coronary vessels showed a dose‐dependent dilation to recombinant (r) IL‐1β that was not affected by an acute increase in temperature of 10 to 20°C. Moreover, the effect of rIL‐1β appears to be endothelium dependent and involve a PG pathway (vessel denudation and Indo both inhibited the rIL‐1β effect). Funding by NSERC & FCT.
Characterization of innate collateral connections in the canine heart
Authors: Spaan Jos , van Horssen Pepijn , van den Wijngaard Jeroen , Siebes Maria
Published: June 2021

Background

Innate coronary collateral connections may be important for recruitment as functional collateral vessels. The aim was to define the distribution of these connections and characterize their diameter and orientation.

Methods

In ex‐vivo canine hearts (n=6) the left main and right coronary artery were cannulated and filled with fluorescent resin (Batson #17). The hearts were prepared for our imaging cryomicrotome, sliced at 20 μm and block face images acquired. The stacks of about 4000 images were mathematically analyzed for vessel segments and pathways. Connections between branching subtrees were detected.

Results

In average 400 connections per heart were found. Diameter distributions were skewed one sided to higher diameters with a mean peak value of 102.27±11.78 μm. In general orientation of these connections are parallel to the epicardium. Additionally, endocardial orientation was preferential parallel towards the longitudinal axis in contrast to midmyocard. The diameter distribution of the segments feeding a closed collateral pathway had a peak diameter of 220 μm. These segments were randomly distributed.

Conclusion

In the dog, pre‐existing collateral connections are randomly distributed in the heart and therefore each coronary artery perfusion territory, large and small, has precursors for collateral vessels outgrowth.

Supported by FP7‐ICT 2007224495 (euHeart)..
Differential Stiffness between Resistance Microvessels and Conduit Arteries in the Coronary Circulation of Ossabaw Swine with Metabolic Syndrome
Authors: Trask Aaron J. , Katz Paige S. , Kelly Amy P. , Cismowski Mary J. , Galantowicz Maarten L. , Neeb Zachary P. , Alloosh Mouhamad , Sturek Michael , Lucchesi Pamela A.
Published: June 2021

The aim of the current study was to determine whether coronary resistance microvessels (CRMs) undergo differential remodeling compared to coronary conduit arteries in metabolic syndrome (MetS). Ossabaw pigs were fed either normal (n=7; Lean) or atherogenic diet (n=7; MetS) for 6 months after which CRM pressure myography and intravascular ultrasound of coronary conduit arteries were performed. MetS CRMs exhibited inward hypertrophic remodeling as shown by reduced luminal diameter and increased wall thickness. Both incremental modulus of elasticity (IME, Lean: 3.6×106 ± 0.7×106 vs. MetS: 1.1×106 ± 0.2×106, p < 0.001) and beta stiffness index (BSI, Lean: 10.3 ± 0.4 vs. MetS: 7.3 ± 1.8, p < 0.001) were decreased in MetS CRMs, while CRM distensibility was augmented (Lean: 0.1 ± 0.004 vs. MetS: 0.18 ± 0.03, p < 0.05). In contrast, BSI was increased (Lean: 4.7 ± 0.8 vs. MetS: 6.7 ± 0.3, p < 0.05) and distensibility was reduced (Lean: 0.36 ± 0.05 vs. MetS: 0.17 ± 0.01, p < 0.01) in MetS coronary conduit arteries. Molecular analysis revealed reduced col1a1/elastin and col3a1/elastin mRNA ratios in MetS CRMs, while these ratios remained unchanged in the coronary conduit arteries. These data suggest that differential remodeling between CRMs and conduit arteries may reflect differential extracellular matrix expression.

Support: NIH T32HL098039 to AJT; NIH HL056046, NCH to PAL; NIH HL062552, UL1 RR025761, Purina TestDiet to MS.
Dopamine D2 receptors regulate the expression of leptin in adipocytes
Authors: Cuevas Santiago , Yang Yu , Armando Ines , Jose Pedro A
Published: June 2021

Leptin is secreted by adipose tissue, regulates food intake, and increases secretion of pro‐inflammatory cytokines. Mouse adipocytes expressed dopamine D2 receptors (D2R). Therefore, we aimed to determine the role of the D2Rs on the regulation of the expression of leptin, adiponectin, and other pro‐inflammatory cytokines in adipocytes. For this experiments mouse 3T3‐L1 cells were cultured to 100% confluency, differentiated to adipocytes for 3 days, and studied 8 days after differentiation. Stimulation of D2Rs with quinpirole (1ìM, 24h) increased the expression of leptin both in cells (23±8%, P<0.05; western blot) and in the media (59±4pg/ml vs. 45±3pg/ml, P<0.05, ELISA), as well as the expression of IL‐6 (49±7%, P<0.05, western blot), but did not modify that of adiponectin. The effects were prevented by the D2R selective antagonist (L741,626). D2R stimulation also increased gene expression (qRT‐PCR) of leptin (1.50±0.03 fold, P<0.05), IL‐6 (2.44±0.08 fold, P<0.05), TNF alpha (2.23±0.06 fold, P<0.05), MCP1 (1.82±0.04 fold, P<0.05), MCP2 (2.43±0.06 fold, P<0.05) and NFkB p50 (1.47±0.02 fold, P<0.05). Adipocyte D2R positively regulates the expression of leptin and pro‐inflammatory cytokines and chemokines in 3T3‐L1. In contrast renal D2R inhibits the their expression. We suggest that the effect of D2R is tissue‐specific but the mechanism involved is undetermined.
Targeted disruption of T‐type calcium channels attenuates NO‐dependent recovery after depolarization
Authors: Hansen Pernille B.L. , Shin Hee-Sup , Skott Ole , Jensen Boye
Published: June 2021

Voltage‐gated calcium channels are involved in the excitation‐contraction mechanism of resistance vessels and play an essential role in regulation of blood pressure. T‐type channels (Cav3.2) are essential for the acetylcholine‐induced relaxation of coronary arteries. We investigated involvement of T‐type channels in agonist‐independent dilatation of perfused mesenteric arteries from two T‐type channel knock‐out (KO) mice strains (Cav3.1 and Cav3.2). Depolarization lead to contraction followed by a secondary dilatation to 51±11% of control diameter (recovery) in wild type (Wt) mice. The recovery was significantly diminished in both Cav3.1 KO and in Cav3.2 KO. Inhibition of eNOS by L‐NAME led to a significantly attenuated recovery in Wt mice. The NO‐donor SNP caused dilatation in preconstricted arteries from both Cav3.1 and Cav3.2 KO mice that was not different from Wt animals. Mean arterial blood pressure and heart rate were measured in conscious mice through chronic indwelling catheters before and after infusion of L‐NAME. NOS inhibition elicited a significant increase in MAP that was sustained after four days in Wt mice (12±2%) whereas no increase was observed in Cav3.2 KO (−3±5%). In conclusion, lack of T‐type channels attenuates NO‐dependent recovery after depolarization in mesenteric resistance vessels and attenuates sustained hypertension after L‐NAME infusion in mouse. The findings are consistent with the notion that endothelial calcium‐influx through T‐type calcium channels stimulates eNOS and NO production in vitro. Our findings in vivo suggest that this mechanism is important for maintaining the systemic tone of NO on the total peripheral resistance.

Research Support: Lundbeck Foundation, The Danish Medical Research Council, The Danish Heart Foundation
Arachidonic acid‐induced dilation in human coronary arterioles: role of endothelial TRPV4‐mediated and membrane potential‐sensitive Ca2+ entry
Authors: Zheng Xiaodong , Nishijima Yoshinori , Gutterman David D. , Zhang David X.
Published: June 2021

Arachidonic acid (AA) induces endothelium‐dependent dilation in human coronary arterioles (HCA) through cytochrome P450 (CYP450) metabolites and K+ channel activation. The precise mechanism by which AA activates endothelial signaling pathways leading to vasodilation remains incompletely understood. Here we investigated the potential role of endothelial TRPV4 in AA‐induced dilation of HCA. In isolated HCA, AA (10−9–10−6 M) caused concentration‐dependent dilation (dilation at 10−6 M, 84 ± 4 %, n=6). This dilation was inhibited by the TRPV4 antagonist RN‐1734 (21 ± 4 %, n=6). Using a fura‐2 Ca2+ assay, AA (3 μM) increased intracellular Ca2+ concentration ([Ca2+]i) in human coronary artery endothelial cells (HCAEC). The [Ca2+]i elevation was inhibited by the TRPV4 antagonists RN‐1734 and HC 067047 and by the CYP450 inhibitors 17‐ODYA and MS‐PPOH. In HCAEC over‐expressed with TRPV4, AA induced an augmented [Ca2+]i increase, which was also inhibited by TRPV4 and CYP450 inhibitors. Interestingly, AA‐induced [Ca2+]i increase was blocked by high K+. By measuring endothelial membrane potential using a fluorescence indicator PMPI, we found that AA induced membrane hyperpolarization, a response not affected by TRPV4 antagonists. Thus, we conclude that AA‐induced dilation of HCA depends on the activity of endothelial TRPV4. AA may activate TRPV4 through CYP450 metabolites and endothelial hyperpolarization.
TRPC1 and TRPV4 are expressed in sensory endings found in regions of venoatrial endocardium where atrial volume receptors are located
Authors: Shenton Fiona , Banks Robert , Pyner Susan
Published: June 2021

Atrial volume receptors (AVRs) detect mechanical deformation as blood returns to the heart. Members of both the ENaC/Degenerin /ASIC and the Transient Receptor Potential (TRP) channel protein families have been implicated in mechanosensation. We have carried out immunohistological investigations to discover potential mechanosensitive ion channels in sensory endings of venoatrial endocardium. Synaptic‐like vesicles, commonly described in mechanosensory endings, are thought to play a key role in regulating afferent output. Double labelling was carried out with a range of anti‐channel antibodies (ENaCβ, ENaCγ, TRPC1, TRPC4/5, TRPC6, TRPV4) together with either anti‐synaptophysin as a vesicle marker or anti‐neurofilament (NF) to identify nerves. TRPC1‐like immunoreactivity (IR) was observed in ganglion cells and nerves of the epicardium, in myocytes; and also in sensory endings of the endocardium, where it was coincident with both synaptopphysin and NF‐IR. Strong TRPV4‐like IR was likewise seen in nerves and ganglion cells. It was coincident with synaptophysin‐IR in both myocardium and epicardium, however it did not appear to stain myocytes themselves. IR was not detected with any of the other anti‐channel antibodies. TRPC1 and TRPV4 channels merit further investigation as potential contributors to mechanosensation in AVRs.

Research supported by EPSRC
Lung endothelial Ca2+ and permeability response to PAF is mediated by TRPC6
Authors: Samapati Rudi , Yang Yang , Yin Jun , Stoerger Christof , Arenz Christoph , Dietrich Alexander , Adam Dieter , Wu Songwei , Freichel Marc , Flockerzi Veit , Uhlig Stefan , Kuebler Wolfgang
Published: June 2021

Platelet activating factor (PAF) increases lung vascular permeability via ceramide formation by acid sphingomyelinase (ASM). Here, we tested the role of endothelial Ca2+ signaling as key regulator of vascular barrier function in this pathway.

In isolated rat and mouse lungs, we quantified endothelial [Ca2+]i by real time imaging, edema formation by lung weight gain, and isolated caveolae to probe for Ca2+ entry channels.

PAF‐induced increases in lung weight gain and endothelial [Ca2+]i were abrogated by inhibitors of ASM or in ASM‐deficient mice, and replicated by exogenous ASM or C2‐ceramide. PAF increased caveolar abundance of TRPC6, which was blocked by ASM inhibition. [Ca2+]i response to PAF was lost in TRPC6 deficient mice, but not in TRPC1,4,5 triple knock‐outs. Activation of TRPC6 replicated the [Ca2+]i and edema response to PAF.

In PAF‐induced lung edema, stimulation of ASM causes recruitment of TRPC6 channels to caveolae allowing for endothelial Ca2+ influx and subsequent hyperpermeability.
Association between depression and smoking in African‐Americans with type 2 diabetes
Authors: Cheema Amanpreet K. , Zarini Gustavo G. , Exebio Joel C. , Ajabshir Sahar , Huffman Fatma G.
Published: June 2021

The purpose of this study was to investigate the association between depression and smoking status in 249 African‐Americans (AA) with (n=129) and without (n=120) type 2 diabetes (T2D). Participants were recruited from multiple sources from Miami‐Dade and Broward Counties, Florida. Socio‐demographic data was collected using a standardized questionnaire. Depressive symptoms were assessed using the Beck Depression Inventory (BDI). Statistical analyses included t‐test, chi‐square, and logistic regression. Adjusted logistic regression showed that individuals with and without T2D who were classified with depression (BDI score ≥ 16 or taking depression medications) were about 2.7 and 3.3 times as likely to smoke than those without depression (OR=2.66, P=.035, 95 CI 1.07–6.59; OR=3.28, P=.022, 95 CI 1.19–9.04 respectively). Findings suggest that depression is likely to play a significant role in the smoking status for AA with and without T2D. Funding for this research was provided through an NIH/NIDDK sponsored grant.
Growth hormone and IGF‐1 deficiency exacerbate high fat diet‐induced endothelial impairment in obese Lewis dwarf rats: implications for vascular aging
Authors: Bailey-Downs Lora C. , Sosnowska Danuta , Toth Peter , Mitschelen Matthew , Gautam Tripti , Henthorn Jim , Ballabh Praveen , Koller Akos , Farley Julie , Sonntag William , Csiszar Anna , Ungvari Zoltan
Published: June 2021

Previous studies suggest that age‐related decline in circulating growth hormone (GH) and IGF‐1 levels significantly contribute to vascular dysfunction in aging by impairing cellular oxidative stress resistance pathways. Obesity in the elderly is increasing at alarming rates and elderly individuals are more vulnerable to deleterious cardiovascular effects of obesity than younger individuals. However, the specific mechanisms through which aging, GH/IGF‐1 deficiency and obesity interact to promote the development of cardiovascular disease remain unclear. To test the hypothesis that low circulating GH/IGF‐1 levels exacerbate pro‐oxidant and pro‐inflammatory vascular effects of obesity, GH/IGF‐ 1 deficient Lewis dwarf rats and control rats were fed a standard diet (SD) or a high fat diet (HFD) for 7 months. HFD‐fed Lewis dwarf rats exhibited an increase in blood glucose levels, lower insulin and impaired glucose tolerance compared to HFD‐fed control rats. Analysis of serum cytokine expression indicated that GH/IGF‐1 deficiency exacerbates HFD‐induced inflammation. GH/IGF‐1 deficiency also exacerbated HFD‐induced endothelial dysfunction, oxidative stress and expression of inflammatory cytokines in aortas of Lewis dwarf rats. Our results suggest that GH/IGF‐1 deficiency renders the cardiovascular system more vulnerable to the deleterious effects of obesity.
Testosterone attenuates metabolic syndrome and coronary artery disease in obese Ossabaw miniature swine
Authors: Alloosh Mouhamad , Buzzitta Kristin M , Sturek Michael
Published: June 2021

Epidemiological studies in humans show a strong inverse association of serum testosterone with metabolic syndrome (MetS) and coronary artery disease (CAD); however, there are no prospective experimental studies. Ossabaw miniature swine develop MetS and CAD when fed excess calorie atherogenic diet. We tested the hypothesis that castrated Ossabaws would develop greater MetS and CAD. Eight castrated and eight intact males (age 6 months) were fed atherogenic diet for 7 months. After 6 months of diet treatment we performed MetS phenotype profiling for adiposity (by computed tomography [CT]), blood pressure, glucose tolerance, total cholesterol, triglycerides, high and low density lipoprotein (HDL and LDL). Intravascular ultrasound (IVUS) imaging quantified CAD. Castrated pigs had more severe MetS than intact pigs, as evidenced by more impaired glucose tolerance, higher total plasma cholesterol, triglycerides, LDL, and LDL/HDL, and less lean mass/greater adiposity. IVUS imaging showed that castrated pigs developed more CAD, including calcification, than intact pigs. We conclude that testosterone attenuated the development of MetS and CAD in obese Ossabaw swine, thus providing an excellent model for study of testosterone therapies. (Support: Purina TestDiet, NIH HL062552)
Ceramide mediates vascular dysfunction in diet‐induced obesity by PP2A‐mediated dephosphorylation of the eNOS‐Akt complex
Authors: Ruan Ting , Ravindran Anindita , Zhang Quan-Jiang , Abel E Dale , Symons J David
Published: June 2021

Our prior studies reveal that pharmacological and genetic inhibition of de novo ceramide synthesis prevents vascular dysfunction in mice following high‐fat feeding and in isolated arteries incubated with palmitate, confirming that ceramide impairs endothelium‐dependent vasorelaxation in a tissue autonomous manner. Mechanisms were pursued in bovine aortic endothelial cells (ECs). De novo ceramide‐induced reductions in agonist‐stimulated eNOS phosphorylation or dimer formation, respectively, were independent of changes in activation of upstream kinases that phosphorylate eNOS (n=12–27), or superoxide anion‐mediated peroxynitrite formation (n=4–18). De novo ceramide biosynthesis increased the association of phosphatase 2A (PP2A) with the eNOS/Akt complex (p<0.05) and paralleled decreased (p<0.05) basal and agonist‐stimulated peNOS( S)1177 (n=12–34). Vascular p‐eNOS(S)1177 was lower (p<0.05), and PP2A association with eNOS was higher (p<0.05), in arteries of obese wild type mice vs. mice with targeted disruption of ceramide synthesis, indicating that similar mechanisms exist in vivo (9–13 mice/group). Moreover, in ECs PP2A attenuates p‐eNOS(S)1177 by preventing phosphorylation of the pool of Akt that co‐localizes with eNOS (p<0.05) and by dephosphorylating eNOS (p<0.05; n=12–18). Ceramide initiates this process by decreasing the association between PP2A and inhibitor 2 of PP2A (p<0.05; n=15–17). Thus ceramide might precipitate obesity‐related vascular dysfunction by a mechanism that involves PP2A‐mediated disruption of the eNOS/Akt signaling complex.
Reduction of Adenylyl Cyclase Type 5 Protects Obesity Induced Cardiomyopathy
Authors: Ho David Wei-Kang , Zhao Xin , Gao Shumin , Tian Yimin , Yan Lin , Vatner Dorothy , Vatner Stephen
Published: June 2021

Since mice with adenylyl cyclase type 5 (AC5) gene disruption eat more than wild type (WT), but weigh less, the goal of this study was to determine if these mice are protected against obesity and the cardiomyopathy induced by a high fat diet (HFD) for 100 days. AC5KO mice gained less weight/kcal consumed than wild type (WT) on HFD, p<0.05, and insulin resistance and glucose tolerance were improved compared to WT on HFD. The HFD induced cardiomyopathy was characterized by reduced, p<0.05, left ventricular (LV) ejection fraction (EF) (53±2.4%) compared with normal diet (ND) (69±2.9%), whereas the reduction in LVEF was less, p<0.05, in AC5KO on HFD (61±3.0%). The cardiomyopathy was also characterized by increased myocardial fibrosis, assessed by picric Sirius red staining, and increased apoptosis, expressed as TUNEL positive cells per mm2. The AC5KO exhibited reduced fibrosis (1.2±0.1%) compared with WT (1.7±0.1%) on HFD, p<0.05, as well as reduced apoptosis (0.5±0.04) compared with WT (0.9±0.1) on HFD, p<0.05. We also examined the effects of pharmacologic inhibition of AC5 with AraAde, an antiviral compound that has AC5 inhibitory effects; weight gain, glucose tolerance, insulin resistance, LV function and fibrosis were all improved compared to vehicle treated mice on HFD. Thus inhibition of AC5 protects against obesity and the cardiomyopathy it induces.
Type 2 diabetes‐induced vascular dysfunction is associated with caveolin‐1 and NADPH oxidase
Authors: Carrillo-Sepulveda Maria Alicia , Goulopoulou Styliani , Matsumoto Takayuki , Hannan Johanna L. , Ergul Adviye , Webb R Clinton
Published: June 2021

Myogenic tone, the ability of the vascular smooth muscle cells to respond to changes in pressure and hemodynamic stress, is augmented in type 2 diabetes; however, the intracellular mechanisms involved remain unclear. Increased levels of vascular reactive oxygen species (ROS) and caveolin‐1 (Cav‐1) have been recently proposed as potential mechanisms for exaggerated myogenic response. However, the sources of ROS as well as the contribution of Cav‐1 in myogenic tone in diabetes are still not known. We hypothesized that increased expression of NADPH oxidase subunits and Cav‐1 are associated with augmented myogenic tone in a rat model of type 2 diabetes. Western blot analysis was used to determine p47 phox subunit and Cav‐1 expression in isolated mesenteric arteries from spontaneously diabetic, non‐obese (Goto‐Kakizaki, GK) and age‐matched (20 weeks old) Wistar rats. Our preliminary data showed increased protein expression of p47 phox subunit (2.5 fold) in GK mesenteric arteries, suggesting that NADPH oxidase may be a potential candidate for ROS‐induced myogenic tone. The levels of Cav‐1, however, were diminished in these vessels (1.1 fold), suggesting that changes in Cav‐1 may be a compensatory mechanism for NADPH oxidase subunit overexpression. Understanding the molecular mechanisms of how ROS are generated is essential for development of novel therapeutic approaches in diabetic vascular dysfunction.
Tuning electrical conduction along endothelial cell tubes via Ca2+‐activated K+ channels
Authors: Behringer Erik , Segal Steven
Published: June 2021

Electrical conduction along coupled cells reflects low axial resistance (ra) of gap junction channels (GJCs) and high plasma membrane resistance (rm) to current flow. Manipulating GJCs alters conducted vasomotor responses but little is known of how conduction may be governed by membrane ion channel activation. We hypothesized that Ca2+‐activated K+ channels (SKCa/IKCa) can tune rm to regulate electrical conduction along endothelial cell (EC) tubes (width, 60 μm; length, ≥ 2 mm) isolated from feed arteries of C57BL/6 mouse skeletal muscle (n = 6–10 males). Using dual simultaneous intracellular microelectrodes, membrane potential (Vm) was uniform along EC tubes (−25 ± 2 mV) and propidium iodide dye injected into one EC spread throughout neighbouring ECs. Conduction amplitude (CA = ΔVm at Site 2/nA current injected at Site 1) decreased with electrode separation distance (CA = 11, 8, 6, 4, 3 ± 1 mV/nA at 50, 500, 1000, 1500, 2000 μm). The SKCa/IKCa activator NS309 (1μM) reduced length constant [λ = (rm/ra)½] from 1360 ± 80 to 840 ± 60 μm (P<0.05). With electrode separation = 500 μm, NS309 from 0.01 to 10 μM increased Vm from −28 ± 1 to −81 ± 1 mV and abolished CA while dye transfer was maintained; blocking SKCa/IKCa (300 nM apamin + 100 nM charybdotoxin) increased CA by ~30% (P<0.05). We conclude that the effectiveness of electrical conduction can be tuned through SKCa/IKCa activation. (NIH R37HL041026, R01HL086483, F32HL110701)
Attenuation of conducted vasodilatation in the skeletal muscle during hyperhomocysteinemia
Authors: Givvimani Srikanth , Narayanan Nithya , Tyagi Neethu , Tyagi Suresh
Published: June 2021

Conduction of vasodilatation (CVD) from distal resistance arterioles to the proximal arterioles and feeding arteries during metabolic demand is mediated by intercellular gap junctions in the vascular endothelium. The role of Hyperhomocysteinemia (HHcy) in musculoskeletal system during CVD is unclear. We hypothesize that during HHcy there is impaired CVD due to decreased expression of endothelial associated connexins and thus decreases tissue perfusion to the contracting skeletal muscles.

Methods

CVD studies are performed in gluteus maximus muscle preparation of wild type (C57BL6/J) and CBS‐/+ (HHcy) mice using intravital microscopy. Expression of connexins and myostatin protein is studied by western blot and Immunohistochemistry methods.

Results

There is decreased CVD and tissue perfusion in response to Acetylcholine in the CBS‐/+ mice compared to wild type controls assessed by intravital microscopy and Laser Doppler. There is decreased expression of connexins and increased myostatin expression in CBS‐/+ mice compared to wild type controls.

Conclusion

Our findings suggest that CVD in skeletal muscle is decreased during hyperhomocysteinemia due to decreased expression of gap junction connexins.

Source of Research support: NIH