bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of circulating globular adiponectin is catalyzed by thrombin
Authors: Wigle Jeffrey Theodore , Tworek Leslee , Taylor Carla G. , Zahradka Peter
Published: June 2021

The circulating levels of the cardiprotective adipokine adiponectin are reduced in obesity and diabetes. Lower adiponectin levels may contribute to the higher incidence of cardiovascular disease in these conditions. A truncated and biologically active form of adiponectin called globular adiponectin is protective against cardiovascular disease. However, little is known about its formation in vivo. Digestion in vitro with trypsin or leukocyte elastase produces a polypeptide with similar structure and biological activity to globular adiponectin. To address how globular adiponectin is produced endogenously, we developed an assay that measures cleavage of recombinant adiponectin into globular adiponectin by serum. N‐terminal sequencing of globular adiponectin revealed proteolysis occurred at Gly‐93. It was also found that the protease exclusively required Ca2+ for activity and that had an apparent molecular mass of about 80 kDa by gel filtration chromatography. The enzyme was insensitive to an inhibitor of leukocyte elastase, but was inhibited by hirudin and argatroban, specific inhibitors of thrombin. Our data indicate that thrombin likely mediates formation of globular adiponectin in the circulation. Supported by Canadian Institutes for Health Research.
Characterization of a neuronostatin receptor in a human kidney cell line
Authors: Elrick Mollisa M , Yosten Gina LC , Samson Willis K
Published: June 2021

Neuronostatin (NST), a recently discovered peptide hormone derived from the somatostatin preprohormone (JBC 283:31949), was shown previously to induce c‐Fos expression in the human gastric cancer cell line (KATOIII). Preliminary data from our lab suggests that the orphan G‐protein coupled receptor, GPR107, is a cognate receptor for neuronostatin. In this study we further explored neuronostatin activity in a human embryonic kidney cell line (HEK 293). GPR107 expression was detected in untreated HEK 293 cells; we therefore hypothesized that HEK 293 cells would respond to treatment with neuronostatin with an increase in c‐Fos expression. Indeed, NST induced a concentration dependent increase in c‐Fos expression after a 60 minute exposure, as determined by real time PCR. These data suggest that HEK 293 cells are a useful model to study neuronostatin signaling. In ongoing experiments, we are attempting to compromise the production of GPR107 in HEK 293 cells using siRNA and, by assaying for responsiveness to neuronostatin, confirm findings in KATOIII cells and in vivo models that have identified GPR107 as the cognate receptor for endogenously produced neuronostatin. Supported by NIJ HL66023.
Negative Regulation of the Human Growth Hormone Gene by Insulin in Primary Pituitary Cell Cultures and a Possible Role for Hypoxia Inducible Factor (HIF)
Authors: Vakili Hana , Jin Yan , Cattini Peter A.
Published: June 2021

Hyperinsulinemia is a key component of metabolic syndrome, and is associated with decreased growth hormone (GH) levels. There is evidence that the human (h) GH gene (hGH1) is a direct target for insulin. A hybrid reporter gene driven by upstream −496/+1 hGH1 sequences, including a putative insulin‐responsive HIF site, was negatively regulated by insulin in transfected tumor cells. Here, we have started to assess whether HIF is associated with insulin regulation of “endogenous” hGH1. In the absence of primary human pituitary cell cultures, transgenic (TG) mice expressing the intact hGH1 locus in a somatotroph‐specific manner were generated. Primary pituitary cell cultures from these TG mice were treated with or without 1–15 nM insulin. A significant decrease in hGH1 RNA levels was detected with a physiological dose of insulin. Stabilization of HIF protein by exposure to the hypoxia mimetic CoCl2 decreased hGH1 RNA levels significantly, and to the same extent as insulin treatment. The effects of inhibition of HIF synthesis by RNA interference, and HIF DNA binding by echinomycin treatment were also pursued. In both cases, the decrease in hGH1 RNA levels seen with insulin treatment was blocked significantly. These data indicate hGH1 RNA levels are regulated negatively by insulin, and that this effect is dependent on HIF protein/DNA binding.
Lack of Testosterone (TST) Worsens Ischemia/Reperfusion– Induced Acute Kidney Injury (I/R‐AKI) and its Deleterious Cardiac Effects
Authors: Soljancic Andrea , Lopez-Ruiz Arnaldo , Chandrashekar Kiran , Maranon Rodrigo , Mahgoub Mohanad , Liu Ruisheng , Juncos Luis A
Published: June 2021

Renal I/R‐AKI results in systemic inflammation, including the heart. We recently reported that plasma TST is reduced during AKI, and its supplementation improves renal injury and inflammation by increasing cytoprotective factors. We now extended this study to test whether the lack of TST exacerbates I/R‐AKI and its cardiac abnormalities. Intact and castrated males (Cast) SD rats were treated with either vehicle or TST (20 ng/kg/min × 10min IV) given 3 hours after subjecting the rats to 40 min of bilateral renal I/R. After 48 hs, we harvested the kidney and the heart, and evaluated renal function and injury (PlCr ‐KIM‐1), cardiac stress (pro‐BNP), and cytoprotective factors (renal and cardiac heme oxygenase‐ HO‐1).

PlCr mg/dl
KIM‐1 pg/ml
BNP ng/ug
Renal HO‐1 ng/ml
Cardiac HO‐1 ng/ml

Sham
0.4 ±0.3
712 ±53
0.18 ±0.02
0.52±0.4
0.4 ±0.06

AKI
1.9 ±1.9*
4576 ±185*
3.8 ±0.1*
5.8±1.2*
5 ±0.1*

AKI‐Cast
2.5 ±2.3&
8972 ±708&
4.4 ±1&
3±0.1&
3.7 ±0.1&

AKI‐Cast‐TST
1.6 ±1#
2348 ±85 #
2.4 ±0.6#
4.4±0.2#
4.6 ±0.1#

Data: Mean±SEM:

p<0.05 vs Sham;

vs AKI;

vs AKI‐CT

Lack of TST exacerbates I/R‐AKI and cardiac stress. Despite the increased injury, HO‐1 is decreased, suggesting that TST may be facilitating HO‐1 expression post AKI.
Pulmonary inflammation and airway hyperresponsiveness associated with placental ischemia induced hypertension in rat dams and offspring
Authors: Johnson Alex C , Eskuri Ryan S , Gilbert Jeffrey S , Regal Jean F
Published: June 2021

Preeclamptic mothers have vascular inflammation with neutrophil infiltration, and post‐partum preeclamptic mothers have increased pulmonary resistance to methacholine i.e. airway hyperresponsiveness (AHR). Since growth restricted offspring of preeclamptic pregnancies are at increased risk for respiratory disease, we hypothesized placental ischemia induced hypertension is associated with neutrophil infiltration into lung and AHR in mother and offspring. On gestation day 14 rats underwent sham surgery or clips were placed on abdominal aorta and ovarian arteries to create reduced uterine perfusion pressure (RUPP) and hypertension by day 19. Dynamic pulmonary resistance in response to increasing doses of methacholine was determined on day 19 using forced oscillation (Scireq). Myeloperoxidase (MPO) in homogenized lungs was assayed as an indicator of neutrophil infiltration. MPO significantly increased in RUPP vs Sham dams (p<0.05) with no difference in AHR. RUPP offspring were growth restricted at birth but not at 8–10 wks of age. Male RUPP offspring, but not female, had increased methacholine response compared to Sham offspring (p<0.05). These data indicate placental ischemia is accompanied by pulmonary inflammation but not AHR in the mother. However, adverse pulmonary reactivity is evident in growth restricted male offspring of a hypertensive pregnancy.

Support: U of M Grant‐in‐Aid & HL109843.
Pravastatin attenuates hypertension and angiogenic imbalance in placental ischemia induced hypertension in the rat
Authors: Bauer Ashley J , Banek Christopher T , Lillegard Kathryn E , Regal Jean F , Gilbert Jeffrey S
Published: June 2021

Preeclampsia is a pregnancy‐specific condition characterized by an imbalance of circulating angiogenic factors and new onset hypertension. Although current treatment options are very limited, recent studies suggest pravastatin may improve angiogenic profile and reduce blood pressure in preeclampsia. We hypothesized pravastatin administration would increase VEGF and reduce arterial pressure (AP) in rats with reduced utero‐placental perfusion pressure (RUPP)‐induced hypertension. On day 14 of pregnancy (21‐term), silver clips were placed on the inferior abdominal aorta and ovarian arteries to generate RUPP‐hypertension. Pravastatin (RUPP+P) was administered i.p. (1 mg/kg/day) through day 19. On day 19 AP was measured via catheter and conceptus data recorded. Blood pressure was increased (P<0.05) in RUPP compared to normal pregnant (NP) dams and pravastatin ameliorated this difference (118±3 vs 91±2 vs 109±2 mmHg). Fetal weight was decreased (P<0.05) in RUPP dams compared to NP controls, but was not improved in RUPP+P (2.07±0.1 vs 2.44±0.05 vs 2.23±0.08 g). RUPP decreased plasma VEGF when compared to NP dams and this was attenuated by pravastatin (759.8±52.3 vs 924.1±43.6 vs 969.5±85.3 pg/mL; P<0.05). Pravastatin did not alter AP, fetal weight, or plasma VEGF in NP dams compared to NP controls. The present data suggest pravastatin may be effective in treating placental ischemia induced hypertension.
Effect of Increased Cortisol on Heart Rate in Pregnant Ewes
Authors: Keller-Wood Maureen , Richards Elaine M , Scheuer Deborah A , Wood Charles E
Published: June 2021

In many species pregnancy alters baroreflex control of heart rate. In ovine, as in human pregnancy, both plasma cortisol and progesterone, hormones influencing the baroreflex, are increased. We tested the hypothesis that cortisol alters baroreflex function in sheep, and this effect is greater in pregnancy. 7 late gestation pregnant (P) ewes and 7 nonpregnant (NP) ewes were studied after stepwise infusions (4d each) of saline (SAL), 1 and 2 mg/kg/d cortisol (CORT1 and CORT2); increases in CORT were within the physiologic range. Blood pressure (BP) and heart rate (HR) were measured by telemetry and baroreflex function was evaluated. Resting BP was significantly greater in NP vs P ewes, but there was no effect of CORT. Pregnancy increased HR, and altered the response to CORT; HR was greater in P vs NP ewes, but increased with CORT2 only in P ewes. Analysis of baroreflex function indicated that CORT increased both minimum and maximum HR in P ewes (CORT2 vs SAL: min HR 72.0±4.3 vs 54.7±4.2; 165.4±9.3 vs 130.8±8.7). The results suggest cortisol increases sympathetic cardiac tone, but that this effect requires other factors occurring in the pregnant state. We found that nitric oxide synthase (NOS) expression in brainstem was altered in pregnancy: in P ewes basal nNOS mRNA was increased (at the level of rostral NTS), whereas iNOS was decreased (level of mid‐NTS), consistent with increased sympathetic tone. Supported by HD056228.
Differential Regulation of Blood Pressure in Transgenic Mice Containing ‐217A/G Polymorphism of Human Angiotensinogen Gene in Response to Immobilization Stress
Authors: Jain Sudhir , Pandey Varunkumar G. , Maharjan Srikrishna , Mopidevi Brahmaraju , Arudra Sri Krishna Chaitanya , Kumar Ashok
Published: June 2021

Stress is a risk factor for hypertension. However, the mechanism by which stress leads to hypertension is unclear. Stress induces glucocorticoid secretion. Increased glucocorticoids trigger DNA binding activity of C/EBPβ and C/EBPδ via glucocorticoid receptors (GR). Here we show that expression of C/EBPβ, C/EBPδ and AGT genes are significantly increased in adrenal medulla, liver and kidney of rats subjected to Immobilization stress (IS).

Renin‐angiotensin system (RAS) plays an important role in regulating blood pressure. Transcription factors C/EBPβ and C/EBPδ are important in regulation of human angiotensinogen (hAGT) gene. We have shown that hAGT gene contains an A/G polymorphism at ‐217, and frequency of ‐217A allele is increased in African‐American hypertensive subjects. Nucleotide sequence of hAGT gene promoter containing ‐217A has homology with the consensus CCAAT/enhancer binding protein (C/EBP) and GR binding site. Therefore, we have generated transgenic mice containing either ‐217A or ‐217G haplotype of the hAGT gene. These transgenic mice were subjected to IS. We found that mice containing haplotype ‐217A subjected to IS has 1.72 fold increased expression of hAGT in liver and 8mmHg increase in blood pressure. Whereas the mice containing haplotype ‐217G shows only 1.2 fold increase in hAGT expression and no significant change in blood pressure. These results suggest that stress leads to the differential regulation of human AGT gene.
Amelioration of Endothelial Dysfunction in Middle Cerebral Arteries (MCA) of Fawn‐Hooded Rats by Antioxidant Treatment and Chromosomal Substitution
Authors: Weinberg Brian , Lombard Julian H
Published: June 2021

The Fawn‐Hooded Hypertensive (FHH) rat is a genetic model of spontaneous high‐renin systemic hypertension and multiple other disorders. As such, it is important to gain an increased understanding of genetic factors controlling systemic vascular reactivity in this strain. We evaluated vascular function in isolated middle cerebral arteries (MCA) from 8–10 week old FHH rats and 3 consomic panels carrying Brown Norway (BN) chromosomes (1,5, and 13) shown to be important in regulating vascular reactivity. All rats were maintained on a low salt (0.4% NaCl) diet since weaning. MCA's from FHH failed to dilate to acetylcholine (ACh, 10−10M to 10−5M). Introgression of BN chromosomes 1 and 13 partially restored ACh‐induced dilation. Introgression of BN chromosome 5 (carrying CYP4A alleles), acute addition of the superoxide (O2• −) scavenger tempol, and scavenging of mitochondrial O2• − with mito‐Q caused a dramatic restoration of ACh‐induced vascular relaxation. Mitochondrial O2• − levels (evaluated with the specific fluorescent probe MitoSOX) were significantly higher in MCA of FHH vs. the well characterized Sprague‐Dawley rat. These findings indicate that chronic elevations in oxidant stress disrupt cerebral vascular reactivity in FHH rats, and that different genes and chromosomes are likely to contribute to vascular dysregulation in different forms of hypertension. (NIH #HL‐65289; #HL‐72920, #HL‐92026).
Analysis of renal hemodynamics and pressure‐natriuresis in Dahl salt‐sensitive rats
Authors: Beard Daniel A
Published: June 2021

Analysis of data on blood flow regulation, filtration, and urine output in Dahl S rats fed on high‐salt (hypertensive) and low‐salt (prehypertensive) diets using simple mathematical models of renal blood flow regulation, glomerular filtration, and solute transport in a nephron reveals the minimal differences between the hypertensive and prehypertensive cases necessary to explain the observed data. It is determined that an increase in renal afferent arterial resistance is able to explain the qualitative differences in observed flows, filtration rates, and glomerular pressure as well as the shift in the pressure‐natriuresis and pressure‐diuresis relationships in animals fed on high compared to low salt diets. Model analysis predicts a decrease in the effective sodium pumping rate in the ascending limb of the loop of Henle in high‐salt fed animals compared to low‐salt fed controls. The finding that a change in afferent arterial tone can explain differences in renal physiological function in high‐salt versus low‐salt fed Dahl S rats suggests the novel hypothesis that, if a shift in the pressure‐natriuresis relationship is the primary cause of elevated arterial pressure in high‐salt fed Dahl S rats, then the primary cause of chronic hypertension in the Dahl S rat is an increase in renal afferent arterial resistance.
Sodium reabsorption in the distal convoluted tubule is enhanced in the 11β‐hydroxysteroid dehydrogenase type 2 knockout mouse
Authors: Hunter Robert William , Mullins Linda , Bailey Matthew A , Mullins John J
Published: June 2021

Mice lacking 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) exhibit hypertension and renal sodium retention that has been attributed to increased activation of the epithelial sodium channel (ENaC) in the connecting tubule and collecting duct. We tested the hypothesis that increased activation of the sodium chloride co‐transporter (NCC) in the distal convoluted tubule (DCT) also contributes to this phenotype.

We compared male 11β‐HSD2 knockout mice at 55 – 65 days of age to wild‐type controls in experiments designed to assess NCC expression and function.

Knockout mice exhibited hyperplasia in the DCT (indentified using an anti‐NCC antibody in immunohistochemistry on formalin‐fixed tissue). NCC protein expression was upregulated in knockout mice (immunoblot of whole kidney homogenates; n = 8, p < 0.01) as was expression of the gene encoding NCC, Slc12a3 (Q‐PCR using whole kidney homogenates; n = 8; p < 0.01). The functional consequences of this were assessed by measuring the effect of an acute hydrochlorothiazide bolus on the fractional excretion of sodium in anaesthetised mice.

Our results suggest that enhanced sodium chloride reabsorption in the DCT contributes to the hypertensive phenotype in this mouse model of apparent mineralocorticoid excess and provide an insight into the role played by adrenal steroids in the regulation of sodium transport in the distal nephron.
H,K‐ATPase type 2 is required for renal adaptation to pregnancy
Authors: Crambert Gilles , Doucet Alain , Salhi Amel
Published: June 2021

Renal reabsorption of K+ during dietary K+ restriction requires the stimulation of H,K‐ATPase type 2 by adrenal progesterone (Elabida et al. 2011, Kidney Int. 80, 256–262). Since renal K+ retention has been observed during pregnancy we investigated whether this pathway could be functional in this physiological state.

During the late part of gestation in mice, the decrease in renal K+ excretion is accompanied by stimulation of HKA2 mRNA and activity. In this condition, kidney function is disconnected from the dietary conditions since gravid mice increase their food consumption (and their K+ intake) by 15%. HKA2‐null mice are unable to efficiently retain K+ during gestation compared to wild‐type littermate (WT) but maintain normal plasma K+ values (around 3.9 mM). This occurs in parallel with a decrease of the fertility rate (86% vs. 25% of successful gestation in WT and HKA2‐null mice, respectively) and a higher mortality rate during gestation in HKA2‐null mice compared to WT. Both phenotypes are reversed when gravid HKA2‐null are given a K+ supplementation in their drinking water. All together, our results provide evidence that pregnancy is a physiological state where kidney is switched from a K+ secretive to a K+ reabsorptive state because of stimulation of H,K‐ATPase type 2. Moreover, inability to do so, have an impact on the development of the gestation.
Renal hemodynamic responses to sustained suppression of central sympathetic outflow
Authors: Iliescu Radu , Georgakopoulos Dimitrios , Irwin Eric D. , Lohmeier Thomas E.
Published: June 2021

Chronic electrical stimulation of the carotid baroreflex causes sustained reductions in blood pressure by suppression of sympathetic activity (SA). The role of SA in controlling renal hemodynamic responses to chronic reductions in renal perfusion pressure is unknown. Therefore, we compared renal and systemic hemodynamics in normotensive dogs in which blood pressure was lowered consecutively for 2 weeks by either baroreflex activation (BA, low SA) or calcium channel blockade with amlodipine, 5mg/kg/day, (CCB, high SA). Both interventions caused a similar sustained reduction in mean arterial pressure (MAP) by ~18 mmHg. Glomerular filtration rate (GFR) responses were as follows:

BA
CCB

Control
67.8±3.3
65.0±2.0

Week 1
59.3±2.4*
62.4±3.0

Week 2
59.7±2.3*
63.7±2.5

Recovery
64.3±2.5
64.2±2.2

P< 0.05 vs control

GFR decreased by 10–15% during baroreflex activation while it was unchanged from control during CCB. Renal blood flow (RBF), measured continuously by ultrasound flow probes, decreased by 10–15% during baroreflex activation while its circadian pattern of variation was maintained. These data are consistent with the hypothesis that despite reduced MAP, sustained suppression of renal SA by BA chronically reduces Na+ reabsorption prior to the macula densa, initiating sustained preglomerular vasoconstriction, which results in lowering of GFR and RBF. As renal disease is commonly associated with hyperfiltration and glomerular hypertension, this effect may provide an additional benefit in hypertensive patients in which reductions in renal hyperfiltration are desirable.
Angiotensin (1‐7) formation in angiotensin converting enzyme 2 knockout mice
Authors: Lino Felipe Luis , Weir Nathan , Grobe Nadja , Elased Khalid , Morris Mariana
Published: June 2021

Angiotensin converting enzyme 2 (ACE2) and its product, angiotensin (1‐7) (Ang (1‐7)), have been shown to protect against renal and cardiovascular dysfunction in animal models of diabetes and hypertension. We found that under baseline conditions, deletion of ACE2 results in a normal mouse phenotype with little or no change in blood pressure and albumin/creatinine excretion in 16 wks old ACE2 knockout (KO) mice compared to wild type (WT) mice. In addition, urinary and renal Ang (1‐7) were not changed suggesting that other factors regulate loss of ACE2. Presence of other peptidases known to form Ang (1‐7), neprilysin (NEP) and angiotensin converting enzyme (ACE), was affirmed using western blot in ACE2KO and WT mice. Activity of NEP and ACE in ACE2KO and WT kidney homogenates was analyzed by mass spectrometry. Results indicate that other proteolytic pathways may be involved in the enzymatic cascade producing Ang (1‐7) thus compensating for ACE2 deficiency. Elucidation of ACE2 independent Ang (1‐7) formation may have clinical implications for treatment of cardiovascular and renal dysfunction. Supported by NIH R01HL093567 and AHA 10PRE4330004 (NMW).
Comparative sugar transport by crustacean hepatopancreas and intestine
Authors: Duka Ada , Ahearn Gregory A.
Published: June 2021

Glucose is transported in crustacean hepatopancreas and intestine by Na+‐dependent cotransport, while Na+‐dependent D‐fructose influx has only been described for the hepatopancreas. In this study lobster (Homarus americanus) hepatopancreas brush border membrane vesicles (BBMV) were used to characterize the cation‐dependency of both 3H‐D‐glucose and 3H‐D‐fructose influxes, while in vitro perfused intestines were employed to determine the nature of cation‐dependent sugar transport in this organ. Over the sodium concentration range of 0–100 mM, both 3H‐D‐glucose and 3H‐D‐fructose influxes (0.1 mM; 1 min uptakes) by hepatopancreatic BBMV were hyperbolic functions of [Na+], exhibiting Km values of 2.30 +/− 0.59 mM (glucose) and 3.79 +/− 1.02 mM (fructose). 3H‐D‐fructose influx by hepatopancreatic BBMV over a potassium concentration range of 15–100 mM was a hyperbolic function of [K+], exhibiting Km value of 12.5 +/− 0.6 mM. Transepithelial 25 μM 3H‐D‐glucose and 3H‐D‐fructose fluxes across lobster intestine over a luminal sodium concentration range of 1 – 50 mM were hyperbolic functions of luminal [Na+], displaying Km values of 6.05 +/− 0.99 mM (glucose) and 10.58 +/− 1.65 mM (fructose). Results suggest that there may be a single carrier process in both organs with a higher affinity for NaCl than for KCl and displays a substrate specificity considerably different than that demonstrated by mammalian SGLT1.
Wogonin reduces infarct size and improves ventricular function in chronic myocardial ischemia rats
Authors: Lin Su-Man , Tsai Shen-Kou , Chih Chun-Lien , Nien Mao-Wei , Huang Shiang-Suo
Published: June 2021

Wogonin (5,7‐dihydroxy‐8‐methoxyflavone), a main active ingredient of Scutellaria baicalensis Georgi (S baicalensis), has been shown to possess antioxidant and anti‐inflammatory effects and to attenuate myocardial ischemia and ischemia‐reperfusion injury. The present study was designed to investigate the action and mechanism of wogonin on ventricular remodeling after chronic myocardial infarction (MI) in rats, which was induced by the permanent ligation of the left anterior descending artery. Rats were randomly allocated to three groups and treated with vehicles or wogonin (0.1 and 0.01 μg/kg, intraperitoneal) once daily for thirty days. We used echocardiography to real time evaluate myocardial function. In addition, We examined the effects of wogonin by hemodynamic studies, histologic examinations, and real‐time quantitative polymerase chain reaction. Rats treated with wogonin (0.1 μg/kg) had significantly increased fractional shortening of the left ventricle, ameliorated left ventricular dilatation, reduced left ventricular end‐diastolic pressure, and reduced mortality and infarct size. Wogonin also attenuated the collagen deposition and the expression of myocardial atrial natriuretic peptide and transforming growth factor‐β1 mRNAs. Overall, our results suggest that wogonin can potentially protect heart from detrimental remodeling.
Protective Role of Coronary Endothelium in the Development of Heart Hypertrophy/Failure
Authors: Sun Xiaowei , Ku David
Published: June 2021

Extensive studies have shown that chronic heart failure/hypertrophy often accompanied by abnormalities in coronary endothelial (CEC) function. In a selective right ventricular hypertrophy (RVH) rat model we noted that the right CEC function was significantly increased prior to the development of RVH, while the left coronaries in the unaffected left ventricle was not altered. As cardiac hypertrophy developed, marked depression of CEC function was noted. To further investigate the significance of CEC function during the development of RVH, we studied the effects of both early (prevention) and late (rescue) treatments with a natural product (garlic) or a standard statin (rosuvastatin, RS). Oral treatment with 1% garlic supplemented diet 3 weeks before, as well as 1 day before or 1 day after the induction of RVH showed significant preservation of CEC function and prevention of RVH. Similar treatments with garlics without its active ingredient, allicin, failed to produce a protective effect. Oral treatment with a water‐soluble HMG‐CoA reductase inhibitor, RS, 1 week before as well as 1 week after the induction of RVH also effectively prevented CEC dysfunction and RVH. Conversely, inhibition of CEC function with L‐NAME treatment exacerbated the development of RVH. Taken together, results of our studies demonstrate that modulation of CEC function provides a novel approach for the management of cardiac hypertrophy.
High fat feeding modulates apoptotic/autophagic pathways in post ischemic myocardium and confers cardioprotection
Authors: Haar Lauren , Ren Xiaoping , Bertaux-Skeirik Nina , Tranter Michael , Rubinstein Jack , Jones WK
Published: June 2021

It is known that chronic consumption of a high fat diet leads to pathology including atherosclerosis, obesity, myocardial infarction, and type II diabetes. However, as suggested by clinical data, we have shown in mice that short‐term exposure to high fat feeding induces cardioprotection against ischemia/reperfusion injury.

We investigated the mechanism for this high fat diet mediated cardioprotection and hypothesize that high fat mediated cardioprotection influences anti‐apoptotic and pro‐autophagic pathways. Our results show that this cardioprotection is NF‐kB dependent and leads to expression level changes in NF‐κB‐dependent genes that contribute to autophagy and apoptosis. Protein levels of Beclin‐1, a key initiator of autophagosome formation, is significantly increased in the ischemic in the post‐ischemic hearts of mice fed a high fat diet relative to control fed mice. In addition, a decrease in TUNEL stained nuclei in sections post‐ischemic of hearts from high fat fed mice relative to control fed suggest a significant decrease in the number of apoptotic nuclei in the ischemic zone of high fat fed mice. The timecourse of high‐fat diet induced autophagy and decreased apoptosis in ischemic hearts correlates with the timecourse of cardioprotection observed as a result of high‐fat feeding. Ongoing work is focused on comparing the autophagic/apoptotic pathway dependence to further discern the mechanistic basis of the protection. Functional studies will be conducted to assess the therapeutic relevance of this effect.

This work was supported by NIH RO1HL091478 (WKJ), NIH T32HL007382 (LH) and Rhen Family Grant (JR)
Glutathione Dependent and Independent Salutary Effects of NAC on HIV Tat Proteinopathy
Authors: Chen Fangping , Lewis William , Hollander John , Baseler Walter , Finkel Mitchell S.
Published: June 2021

We have previously reported that cardiac myocyte‐specific expression of HIV‐Tat (Tat) results in a murine cardiomyopathy model. We now report that cardiac myocytes isolated from Tat exhibit decreased ATP (p<0.01; n=8–9) and GSH levels (p<0.01; n=8–9), decreased GSH/GSSG ratio (p<0.01; n=8–9), increased H2O2 (p<0.05; n=6) and catalase (p<0.05; n=6), and GPX1 (glutathione peroxidase 1) activities (p<0.05; n=6), blunted systolic function (%Fractional shortening, p<0.01; n=15; +dl/dt p<0.01; n=15) and diastolic function (‐dl/dt, p<0.01; n=15), and inotropic responses to Ca++ (p<0.01, n= 15) and shortened anatomical and functional survival in vitro (p<0.01, n=15). Treatment of Tat cardiac myocytes with the sulfhydryl donor, N‐acetylcysteine (NAC; 10−4 M) reversed both the positive and negative inotropic defects in Tat; increased GSH (p<0.01); GSH/GSSG (p<0.01); reversed H2O2 level (p<0.05) and GPX1 activity (p<0.05); normalized the blunted inotropic response to Ca2+ (p<0.01). In addition, NAC 10−7 M normalized duration of contractile function from <40 min to >120 min (p<0.01), while having no affect on GSH, GSH/GSSG. Elucidating molecular mechanisms involved in the GSH‐dependent and GSH‐independent salutary effects of NAC could identify novel therapeutic targets to mitigate the pathogenic effects of proteinopthies in cardiomyopathies and heart failure.
Inhibition of Protein Kinase β2 Attenuates NOS Uncoupling and Myocardial Dysfunction in Streptozotocin‐Induced Diabetic Rats
Authors: Lei Shaoqing , Li Haobo , Wang Tingting , Liu Yanan , Mao Xiaowen , Hei Ziqing , Irwin Michael G , Xia Zhengyuan
Published: June 2021

Hyperglycemia induced protein kinase C (PKC) β activation is implicated in diabetic cardiomyopathy, which is also associated with impaired function of endothelial nitric oxide (NO) synthase (eNOS). We postulated that PKCβ2 inhibition may attenuate myocardial dysfunction by rescuing eNOS function and reducing eNOS uncoupling in streptozotocin (STZ)‐induced diabetes. Control (C) or STZ‐induced diabetic (D) rats were treated or untreated with PKCβ inhibitor LY333531 (LY, 1 mg/kg/day) for four weeks. The ratio of peak velocity of early and late diastolic filling (E/A) measured by echocardiography was decreased, while left ventricular isovolumic relaxation time (IVRT) and the heart to body weight ratio were increased in D rats. All of these changes were attenuated by LY (P<0.05 D vs. C). Cardiac O2− and nitrotyrosine production were increased, whereas NO were decreased in D rats. Diabetes induced increase of O2− levels was blocked by the NOS inhibitor L‐NAME, indicating “NOS uncoupling”. In addition, cardiac iNOS expression was increased, while levels of eNOS, p‐eNOS (Ser 1177) and p‐Akt (Ser 473) expression were decreased in D rats. All the alterations were attenuated or reversed by LY. We conclude that PKCβ2 inhibition may attenuate myocardrial dysfunction in diabetes by restoring Akt‐dependent eNOS/NO signaling and maintaining eNOS coupling.

Supported by RGC/GRF (782910, 766709)
Viscous dietary fibers added to a high fat diet decrease adiposity, improve glucose control and alter fuel utilization in obese rats
Authors: Brockman David A. , Chen Xiaoli , Gallaher Daniel D.
Published: June 2021

Viscous dietary fibers reduce the postprandial rise in blood glucose and decrease adiposity in normal and diabetic rats. The purpose of this study was to examine the effects of viscous fibers on fat mass and fuel utilization in a model of diet‐induced obesity. Wistar rats were fed a high fat diet (60% energy from fat) containing 5% fiber as either cellulose (non‐viscous) or one of two highly viscous fibers ‐ hydroxypropyl methylcellulose (HPMC) or guar gum. After 10 weeks of feeding, the HPMC and guar groups had lower fat pad weights and total body fat mass compared to the cellulose group. The guar group had a decreased glucose tolerance test area under the curve (AUC) indicating improved glucose control. AUC during a meal tolerance test was not different with viscous fiber treatment. However, after 3 weeks of feeding, the respiratory quotient (RQ) AUC over 24 hours was increased with HPMC and guar compared to cellulose, implying greater carbohydrate oxidation. The RQ AUC during fasting did not differ between the HPMC and cellulose groups but after a meal it was greater in the HPMC and guar groups. This suggests a quicker transition from fatty acid to carbohydrate oxidation, possibly through decreased insulin resistance. These results indicate that consumption of viscous fibers with a high fat diet can improve glucose control, lower adiposity and increase fuel flexibility.

Grant Funding Source: MN Agricultural Experiment Station, MN Obesity Consortium
Discovery of Early Life Plasma Protein Signatures for Asthma Development
Authors: Xu Haili , Radabaugh Timothy , Lu Zhenqiang , Billheimer Dean , Vercelli Donata , Halonen Marilyn , Lau Serrine S
Published: June 2021

Objective

The objective of our study was to identify plasma proteomic signatures that accompany asthma risk in early life using an antibody array approach.

Methods

Blood plasma samples from 2 groups of three‐year‐old subjects enrolled in the Tucson Infant Immune Study were assayed for relative expression of 507 proteins by RayBio's Biotin Label‐based Human Antibody Arrays. The 2 groups were subjects with no asthma by age 9 or wheeze at age 9 (no asthma/wheeze; n=6) and subjects with asthma by age 9 and wheeze at age 9 (asthma/wheeze; n=11). The intensity values of signals from each plate were ranked. Rank differences between the 2 groups were evaluated for each protein using the t‐test. Proteins showing significant group differences were identified by a false discovery rate (FDR) p value of<0.1.

Results

Two proteins were found to be substantially altered in rank among subjects with asthma/wheeze versus subjects with no asthma/wheeze. The rank of eotaxin‐3 was increased (pFDR = 0.0598) and the rank of erythropoietin was decreased (pFDR =0.0282) in subjects with asthma/wheeze.

Conclusion

Our study suggests that the relative expression of proteins eotaxin‐3 and erythropoietin in plasma can serve as candidate biomarkers differentiating subjects with from those without asthma/wheeze.

Funded by ABCD Pilot Project, P30ES006694 and ES018328
Assessment of safety and embryotoxicity of (−)‐Epigallocatechin‐3‐gallate
Authors: Rodríguez-Fragoso Lourdes , Gonsaga-Morales Ana Isabel , Esparza Jorge Reyes
Published: June 2021

The beneficial effects of green tea are attributed primarily to (−)‐Epigallocatechin‐3‐gallate (EGCG). However, it has been studied very little about their toxicity when administered at high doses or as a pure compound. Because of this, it is important to assess safety. We used human tumor and no tumor cell lines, we assessed the viability and proliferation by MTT assay, identifies the type of cell death and alterations in the cell cycle by flow cytometry. We evaluated the antifungal effect in yeast (C. albicans 10231, C. albicans 752 and C. krussei). Embryotoxic effect was evaluated on chicken embryos. 293‐Q cell proliferation decreased from 100 μM, produced death by necrosis and decreased G0/G1 phase from 1 μM. WRL‐68 cells, increased viability from 100 μM, decreased proliferation, altered S phase and apoptotic death occurred only at 1000 μM. Produced an inhibition on the growth of C. 10231 and C. krussei but not on C. albicans 752. It produced no embryotoxic effects. Our conclusion is that the use of EGCG is safe at concentrations below 100μM and is not embryotoxic.
Effects of menopausal hormone therapy (MHT) on vascular reactive molecules in platelets
Authors: Lieser Elizabeth Ann , Miller Virginia M , Jayachandran Muthuvel
Published: June 2021

Activated platelets release mitogenic factors, vascular remodeling enzymes, inflammatory and cellular adhesive molecules into the vascular microenvironment to alter chemotactic and adhesive properties of vascular endothelium. Platelet‐derived growth factor‐BB (PDGF‐BB) and matrix metalloproteinase‐2 (MMP‐2) increased in the platelets following ovariectomy but decreased with estrogen treatment in pigs. Experiments were designed to identify changes in these factors using custom antibody arrays in lysate of platelets collected from women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic. Washed platelets were prepared from anticoagulated blood before (baseline) and 48 months after randomization into either placebo or oral or transdermal MHT. Preliminary analysis shows after 48 months compared to baseline, platelet content of C‐reactive protein, intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), PDGF‐BB, MMP‐2 and transforming growth factor‐beta (TGF‐beta) were lower (20–60%) in two groups but higher (20–74%) in one group. Based on our studies in pigs, it is expected that the two groups showing decreases in platelet proteins would be from women randomized to MHT. However, KEEPS will not be unblinded until February 2012. (Supported by grants from Aurora Foundation, NIH HL90639, 1UL1 RR024150).
The Effects on Thrombin Generation Following Resuscitation with Lactated Ringers and Fresh Frozen Plasma
Authors: Pawelczyk Nicholas S , Huby Maria , Baer Lisa A , Salsbury John R , Matijevic Nena , Wang Willa , Cotton Bryan A , Holcomb John B , Wade Charles E
Published: June 2021

Following trauma, patients are infused with fluids such as crystalloids or fresh frozen plasma. Observing thrombin generation is key to demonstrating changes in hemostatic potential due to the infusions. Our objective was to explore the effects of resuscitative fluids on coagulation as measured by Calibrated Automated Thrombography (CAT). Male rats underwent laparotomy with gut externalization and hemorrhagic shock to MAP of 35mmHg until decompensation (inability to auto‐resuscitate). Rats were randomized based on fluids received: Lactated Ringers (LR; n=7) and Fresh Frozen Plasma Day 0 (PL‐0; n=8). PL‐0 was obtained by pooling citrated rat plasma from non‐experimental donor rats and PL‐0 coagulation factors were not significantly different from baseline. Prior to resuscitation, there were no significant differences in coagulation factors between LR and PL‐0. Resuscitation with LR resulted in a significant decrease in lagtime but maintenance of high endogenous thrombin potential (ETP) and peak levels. Resuscitation with PL‐0 resulted in a significant increase in lagtime, ETP, and Peak. Following trauma and hemorrhagic shock, resuscitation with LR produced a prothrombotic state while resuscitation with PL‐0 produced a coagulopathic state. Hemostatic potential is important in determining which fluid may be required during trauma management. (UT Health Science Center–Houston CeTIR Dept Grant).
Effect and mechanism of action of mucosal leptin on glucose absorption and Na+/K+ ATPase in Caco‐2 cells
Authors: El-Zein Ola Mohammad , Kreydiyyeh Sawsan Ibrahim
Published: June 2021

The effect of mucosal leptin on glucose intestinal absorption and on the Na+/K+ ATPase was investigated using Caco‐2 cells as a model. Cells were cultured on transwell permeable supports and treated with leptin 23 days after confluence. Leptin, [14C] 3‐O‐methyl‐ D‐glucose and [3H] mannitol were added to the upper well, and cells were incubated at 37°C for 50 min. Samples were collected from the lower chamber every 10 min and assayed for radioactivity. Leptin reduced significantly glucose absorption. This reduction was mediated via p38 mitogen‐activated protein kinase (p38MAPK), extracellular signal‐regulated kinase, phosphatidylinositol‐3 kinase, and protein kinase C (PKC) since the effect of leptin disappeared when each of the kinases was inhibited individually. Because glucose uptake is dependent on the Na+ gradient established by the Na+/K+ ATPase, the pump itself may be targeted by leptin. To test this hypothesis, the activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of enzyme inhibitors, and in presence of inhibitors of the different kinases studied above. The activity of the Na+/K+ ATPase was also reduced significantly by mucosal leptin through an activation of p38MAPK and an inhibition of PKC. It was concluded that the effect of mucosal leptin on glucose absorption is due, at least partially, to a lower activity of the Na+/K+ ATPase. Supported by URB.
Whole body C‐cbl Associated Protein (CAP) deleted mice display impaired endothelium dependent dilation and nitric oxide bioavailability
Authors: Henson Grant David , McCullagh Joseph P , Donato Anthony J , Lesniewski Lisa A
Published: June 2021

In endothelial cells, high glucose impairs insulin‐stimulated endothelial nitric oxide synthase (eNOS) activity in a c‐casitas B lymphoma (c‐cbl) associated protein (CAP)‐dependent manner. In adipocytes, the CAP/cbl complex associates with lipid rafts and participates in GLUT4 trafficking upon insulin stimulation. Together, these findings raise the possibility that CAP/cbl participate in eNOS activation/trafficking. We tested the hypothesis that compared to wild‐type (CAP+/+) littermates, arterial function is impaired in mice with whole body CAP deletion (CAP−/−). Maximal ex vivo carotid artery endothelium dependent dilation (EDD) to acetylcholine (ACh) was reduced in CAP−/− versus CAP+/+ (72.2 ± 7.3 v 99.84 ± 0.8%, p < 0.05). Impaired EDD was associated with reduced nitric oxide (NO), evidenced by the abolition of differences in EDD after incubation with a NOS inhibitor. This does not appear to be associated with increased superoxide, as incubation with a superoxide dismutase mimetic failed to improve dilation in CAP−/− mice. Endothelium independent dilation was not different between groups. CAP deletion was without effect on aortic stiffness (pulse wave velocity) or mean arterial pressure (p's > 0.19). CAP deletion results in impaired EDD via a superoxide‐independent reduction in NO. Although the mechanism is unknown, dysregulation of eNOS trafficking is a possibility requiring examination.
Effect of pulsatile shear stress on endothelial cell activation
Authors: Shanmugavelayudam Saravan Kumar , Rubenstein David Alan , Yin Wei
Published: June 2021

Localization and pathogenesis of coronary heart disease (CHD) is closely related to the alterations in local blood flow patterns and corresponding wall shear stress (WSS) distribution. Endothelial cells (EC), lining the innermost layer of blood vessels, are susceptible to altered WSS and may promote atherosclerotic lesion initiation. The goal of the present study was to investigate the effect of physiologically relevant dynamic shear stress on EC activation and injury. Based on a 3D computational fluid dynamics model, pulsatile WSS waveforms representing normal, recirculation (low amplitude unidirectional and oscillatory) and stenosis (elevated) shear conditions were applied to coronary artery endothelial cells in a cone and plate shearing device, and EC response was measured at protein and RNA levels, using ELISA and real time PCR. Proteins examined include intercellular adhesion molecule – 1 (ICAM‐1), tissue factor (TF), thrombomodulin (TM) and von Willerbrand factor (vWF). The results demonstrated that, at protein level, ICAM‐1 and TF expression was upregulated under recirculation shear; TM expression was downregulated under stenosis shear; while no significant change was observed with vWF expression. Similar changes were observed at the RNA level as well. These results suggest that altered WSS had a significant role on EC activation and the growth of atherosclerotic lesion. Funding Source : American Heart Association
The endothelial cells of ductus arteriosus have a unique gene profile to control vascular morphology
Authors: Liu Norika , Yokota Tomohiro , Maekawa Shun , Yokoyama Utako , Kato Takashi , Minamisawa Susumu
Published: June 2021

Patency of ductus arteriosus (PDA) is morbid; especially it is a severe problem in preterm infants. Hence, to investigate the mechanism of DA closure is important for pediatrics. The present study focused on endothelial cells (ECs) in the DA because ECs lining vessels have a variety of functions and play a central role in homeostasis of the circulatory system. We hypothesized that ECs of the DA had unique characteristics from other vessels and that they should be the primary target to investigate the molecular mechanisms of the DA‐specific vasoconstriction and vascular remodeling.

Using fluorescence activated cell sorter, we isolated pure ECs from pooled tissues from the DA or the aorta of Wistar rat fetuses on the 21st day of gestation (e21) or 30 minutes after birth (day0). The gene profiling of those sorted cells were then analyzed by DNA microarray (GeneChip Rat Gene 1.0 ST Array, Affymetrix). We found 61 genes in e21 and 63 genes in day0 were specifically expressed in DA ECs than in aortic ECs (the data were cut off by p value<0.01 or fold change > 2.0). Among them, the genes related to cell adhesion and blood vessel morphogenesis development, such as Vegf‐a, Tgf‐beta, Tbx1, Fgf10 and Pitx2 were abundant in DA ECs, which indicates those genes play an important role in specification of DA morphology and function.
Measurement of the Binding Strength of Endothelial Progenitor Cells to Vascular Endothelial Cells in Shear
Authors: Prisco Anthony , Greene Andrew
Published: June 2021

Endotheial Progenitor Cells (EPCs) from the bone marrow participate in angiogenesis. When isolated, cultured and injected intravenously into an angiogenesis incompetent strain, EPCs are capable of restoring angiogenesis. One of the key factors in this restoration is the binding dynamics of the EPC‐endothelial cell (EC) interaction. When treated with TNF‐[alpha], endothelial cells are “activated,” through the upregulation of integrins on the cell surface which contribute to the EPC‐EC interaction. We built a custom in‐vitro flow chamber that exerts a shear stress gradient on cultured cells to determine the extent that EPCs derived from 8 week Sprague Dawley (SD) rats were bound to ECs (3 hour binding period) derived from 1 day SD neonates (Cell Biologics) with and without EC treatment (TNF‐[alpha] (1ng/mL) for 12 hours). We then used the chamber to measure the strength of the interaction. To investigate the mechanism of EPC‐EC binding, we compared the surface protein expression of the ECs treated with TNF‐[alpha], to vehicle treated cells. Using a targeted plasma membrane proteome capture followed by tandem mass spectrometry analysis, differentially expressed membrane proteins revealed several candidates for the EPC‐EC interaction.

Supported by NIH082798