bluuebunny/crossref_metadata_2025 huggingface dataset:
HIV Tat induces expression of ICAM‐1 in human umbilical vein endothelial cells: Implications for HIV‐associated cardiomyopathy
Authors: Yao Honghong , Duan Ming , Lund Amie K , Buch Shilpa
Published: June 2021

Cardiac involvement is a well‐documented complication of human immunodeficiency virus (HIV) infection. HIV transactivating protein, Tat, plays a key role in activating endothelial cells. In the present study we demonstrate that exposure of human umbilical vein endothelial cells (HUVECs) to HIV Tat induced the expression of cell adhesion molecules specifically, ICAM, leading to increased monocyte adhesion to the endothelial cells. This effect of Tat was mediated through activation of mitogen‐activated protein kinases and downstream NF‐êB. Increased ICAM was regulated by miR‐221 and to some extent mir‐222 both of which are known to target ICAM. Functional inhibition of the respective miRs with anti‐miRs resulted in induction of the ICAM protein in HUVECs. Furthermore, Tat‐stimulated regulation of ICAM via miR221/222 involved the NF‐kB‐dependent pathway. Functional implication and specificity of up‐regulated ICAM was confirmed using the ICAM neutralizing antibody in vitro in cell adhesion assays. These findings were also confirmed in vivo using the HIV transgenic rat that demonstrated increased expression of ICAM in the aorta and endothelial cells of the heart. Taken together, these findings implicate that Tat‐mediated induction of ICAM expression may play a critical role in monocyte adhesion observed in HIV‐associated cardiomyopathies.
Reduced RhoA activity mediates the acute alcohol intoxication‐induced reduction of lymphatic myogenic constriction independently of cytosolic [Ca2+]
Authors: Souza-Smith Flavia Moreira , Kurtz Kristine K , Breslin Jerome W
Published: June 2021

The RhoA‐ROCK pathway is involved in [Ca2+]‐independent regulation of MLC20 phosphorylation through its inhibition of MLCP and, thus, the regulation of smooth muscle contraction. We previously showed that alcohol modulates the intrinsic contractile cycle of mesenteric lymphatics that provides the driving force for lymph flow. We hypothesized that either calcium signaling or sensitivity mediate the changes in intrinsic lymphatic pumping caused by alcohol. Acute alcohol intoxication was produced by intragastric administration of 30% alcohol to conscious, unrestrained rats through surgically implanted catheter. Isovolumic administration of water (vehicle) served as control. Mesenteric lymphatics were isolated, cannulated and loaded with Fura‐2 AM. Calcium measurements were performed at a basal intraluminal pressure of 2 cm H2O and during pressure steps to 4, 6, 8, 10 and 12 cm H2O. RhoA activity was determined using a G‐LISA™ assay. With each pressure step [Ca2+] gradually increased for both control and alcohol, however myogenic constriction was impaired in the alcohol group. RhoA activity was significantly reduced in lymphatics from the alcohol group compared with controls. Reduced myogenic constriction from alcohol treated lymphatics is due to reduction on [Ca2+] sensitivity likely mediated by RhoA signaling.
Reactive Oxygen Species (ROS) from NADPH and Xanthine Oxidase Modulate the Cutaneous Local Heating Response in Chronic Fatigue Syndrome
Authors: Medow Marvin Scott , Aggarwal Arun , Baugham Ila , Messer Zachary , Stewart Julian M.
Published: June 2021

Local cutaneous heating causes vasodilation as an initial peak, a nadir, and increase to plateau. We showed reactive oxygen species (ROS) produced by Ang‐II via NADPH and xanthine oxidase, modulates this response in healthy controls. The initial peak, due to C‐fibre nociceptor‐mediated axon reflex and local release of calcitonin gene‐related peptide, substance P and neuropeptide Y, is blunted with local anesthetics. To determine the role of ROS in this neurovascular response, we studied orthostatically‐challenged, Chronic Fatigue Syndrome (CFS) subjects. We heated the skin of 9 CFS subjects (15.8–22.0 years) to 42°C and measured local blood flow. While CFS subjects had lower baseline blood flow (79% of control), there were no differences between groups to local heat. We then measured CFS subjects while perfusing apocynin (NADPH oxidase inhibitor), allopurinol (xanthine oxidase inhibitor), tempol (superoxide inhibitor), and ebselen to reduce H2O2 via microdialysis catheters. Allopurinol and ebselen only enhanced the first heat peak (117% and 126% of heat‐alone, respectively). Heat with apocynin caused an increase in baseline (170% of non‐drug) and of the first heat peak (114% of heat‐alone). Tempol had no effect. Thus the response to heat, as indicated by the first peak, may be altered by local levels of ROS in CFS subjects, and may be related to hyperesthesia/hyperalgesia reported by many individuals with CFS. Supported by the National Heart, Lung, and Blood Institute Grants 1‐RO1‐HL‐074873, 1‐RO1‐HL‐087803 and 1‐F30‐HL‐097380 and a grant from the Chronic Fatigue and Immune Dysfunction Syndrome Association.
Tyrosine nitration of voltage dependent anion channels induced by peroxynitrite alters protein structure and function in vitro
Authors: Yang Meiying , Camara Amadou K.S. , Kwok Wai-Meng , Zhou Yifan , Gadicherla Ashish K. , Stowe David F.
Published: June 2021

Voltage dependent anion channels (VDAC) are key and abundant outer mitochondrial membrane (OMM) proteins. Peroxynitrite (ONOO−) can induce irreversible tyrosine nitration (Ntyr) of some proteins to alter their structure and function. We showed previously that Ntyr of VDAC occurs in cardiac ischemia and reperfusion (IR) injury due to excess superoxide (O2 •−) reacting with nitric oxide to form endogenous ONOO−. We hypothesized that ONOO− directly induces VDAC specific Ntyr and alters VDAC structure and channel activity. To test this, we exposed recombinant VDAC (rVDAC; rat heart VDAC‐1 full‐length cDNA, GenBank: BC072484, Open Biosystems) to exogenous ONOO− in vitro. We evaluated VDAC Ntyr by Western blot with 3 nitrotyrosine (3‐NT) antibody and examined for sites of Ntyr by MALDI‐TOF MS assay. We found that rVDAC can be Ntyr by as low as 10 μM ONOO−, with a higher band intensity as [ONOO−] is elevated and also that Ntyr of rVDAC induced formation of dimers and trimers. The MS assay showed nitration occurred at either 62Y or 67Y. We monitored channel activity of rVDAC by incorporating rVDAC into planar lipid bilayers. At a test potential of −10 mV, we found that 50 μM ONOO− enhanced rVDAC channel conductance as shown by current amplitudes of −25 pA and −30 pA compared to −18 pA before adding ONOO−. Our results demonstrate that rVDAC can be nitrated by ONOO−, which induces both structural and functional changes in rVDAC.
Diabetes induces endothelial dysfunction: role of eNOS uncoupling and arginase
Authors: Thengchaisri Naris , Hein Travis W. , Kuo Lih
Published: June 2021

We studied the role of arginase and eNOS in vascular dysfunction using porcine diabetic model. Pigs (8‐wk old) were subjected to streptozotocin (STZ) injection for diabetes induction. Two weeks after STZ or saline (control) injections, coronary arterioles (50–100 μm) were isolated and pressurized for in vitro studies. The vasodilations to eNOS activators (flow and serotonin), but not to NO donor sodium nitroprusside, were significantly reduced in the diabetic group. Application of H2O2 scavenger catalase (1,000 units/ml) did not alter the serotonin response in control or diabetic group but inhibited flow‐mediated dilation in vessels from the diabetic pigs. The inhibitory effect of catalase on flow‐mediated dilation was absent after extraluminal application of L‐arginine (3 mM). Furthermore, inhibition of NOS with L‐NAME (10 μM) completely abolished serotonin‐ and flow‐mediated dilations in both control and diabetic groups. Interestingly, treating the diabetic vessels with arginase inhibitor norNOHA (0.1 mM) improved the serotonin‐induced vasodilation to the same extent as by L‐arginine application. In conclusion, H2O2 from the uncoupled eNOS serves as a vasodilatory mediator for flow‐induced dilation of diabetic porcine coronary arterioles. On the other hand, L‐arginine deficiency due to arginase upregulation contributes to the impaired coronary arteriolar dilation to serotonin in diabetes.
Ischemia‐induced mobilization of lysosomal iron predisposes rat hepatocytes to ischemia‐reperfusion (IR) injury
Authors: Zhang Xun , Schwartz Justin , Lemasters John
Published: June 2021

Reactive oxygen species (ROS) initiate onset of the mitochondrial permeability transition (MPT) and play a key role in IR injury. Iron is a critical catalyst for ROS generation. Accordingly, our aim was to investigate the role of chelatable iron in ROS‐dependent MPT formation and cell death during IR to rat hepatocytes. Cells were incubated anoxically at pH 6.2 for 4 h and re‐oxygenated at pH 7.4 to simulate IR. Chelatable iron, ROS, mitochondrial membrane potential and cell death were monitored by confocal imaging of calcein, chloromethyl dichlorofluorescein, tetramethylrhodamine methyl ester and propidium iodide, respectively. Ischemia caused progressive quenching of cytosolic calcein, indicating an increase of chelatable Fe2+, which was suppressed by desferal and starch‐desferal pretreatment. Quenching of calcein was also blocked by ebselen and ferristatin, inhibitors of lysosomal divalent metal transporter‐1. Ischemia induced quenching of calcein loaded into mitochondria, which was blocked by desferal, starch‐desferal and Ru360. These agents also decreased mitochondrial ROS formation, MPT onset and cell killing after reperfusion. In conclusion, lysosomal iron is mobilized into mitochondria during ischemia. Increased mitochondrial iron then predisposes cells for ROS‐mediated MPT opening and cell killing after reperfusion. (Supported by NIH grants DK073336 and DK37034)
Blood cell‐associated angiotensin II type‐1 receptors and gp91phox mediate the inflammatory and thrombogenic responses elicited by chronic angiotensin II administration
Authors: Yildirim Alper , Granger D. Neil
Published: June 2021

Angiotensin II (AngII) contributes to the pathogenesis of hypertension and other cardiovascular diseases. AngII induces a pro‐oxidative, pro‐inflammatory, and pro‐thrombogenic phenotype in vascular endothelial cells. Although the peptide promotes the recruitment of leukocytes and platelets and induces oxidative stress in the microvasculature, the mechanisms that underlie these responses remain poorly defined. In this study, we addressed the contributions of AngII type‐1 receptors (AT1r), and gp91phox to the recruitment of leukocytes and platelets and production of reactive oxygen species (ROS) in skeletal muscle venules during chronic (2 wks) infusion of AngII in wild type (WT) and mutant mice. Intravital video microscopy was used to measure the adhesion and emigration of leukocytes, the adhesion of fluorescently labeled platelets, and dihydrorhodamine oxidation (a measure of oxidative stress) in cremaster muscle post‐capillary venules. In WT mice, AngII infusion induced the adhesion of leukocytes and platelets and enhanced ROS production in the vessel wall. These changes in blood cell adhesion and ROS production were not observed in AT1r−/− mice, in AT1r−/− bone marrow chimeras (blood cells deficient in AT1r), gp91phox−/−, gp91phox−/− chimeras (blood cells deficient in gp91phox) and in WT mice rendered neutropenic via i.p injection of anti‐mouse Gr‐1 antibody. Thrombocytopenic WT mice (platelets depleted by i.p. injection of rabbit anti‐mouse thrombocyte antiserum) responded similar to WT mice. These findings implicate blood cell‐associated AT1r and gp91phox in the induction of the proinflammatory and prothrombogenic phenotype in microvessels exposed to chronic AngII. (Supported by HL26441)
Effect of N‐Acetyl‐Seryl‐Aspartyl‐Lysyl‐Proline (Ac‐SDKP) on intercellular adhesion molecule 1 (ICAM‐1) and proinflammatory cytokines and chemokines in experimental autoimmune myocarditis (EAM)
Authors: Nakagawa Pablo , Rhaleb Nour-Eddine , Chen Xiaojuan , Liao Tang- Dong , Smolarek Derek M. , Yang Xiao-Ping , Liu Yun-He , Carretero Oscar A.
Published: June 2021

The naturally occurring tetrapeptide Ac‐SDKP improves cardiac function in EAM. We hypothesize that in EAM, Ac‐SDKP interferes with leukocyte infiltration and this is mediated by the reduction of ICAM‐1, cytokine and chemokine expression. EAM was induced in rats by porcine cardiac myosin and treated with vehicle or Ac‐SDKP (800 μg/kg/day). Cardiac inflammation was graded according to a four‐tier system. Infiltration of macrophage and T helper cells were quantified by immunohistochemistry. ICAM‐1 was detected by Western blot. Chemokines and cytokines were quantified using a proteomic cytokine array. Ac‐SDKP reduced cardiac inflammation (EAM: 2.2 ± 0.2 A.U. vs. EAM + Ac‐SDKP: 1.5 ± 0.2**A.U.), infiltration of macrophages (257 ± 37 vs.138 ± 9* ED‐1 cells/mm2) and T helpers (148 ± 20 vs. 97 ± 17* CD4 cells/mm2), ICAM‐1 (2.918 ± 0.880 vs. 0.012 ± 0.002** A.U.), interleukin1α (IL‐1α )(1.6 ± 0.4 vs. 0.1 ± 0.1**A.U.), and cytokine‐inducible neutrophil chemoattractant (CINC‐1) (1.2 ± 0.2 vs.0.2 ± 0.1*A.U.). In conclusion, Ac‐SDKP prevents leukocyte infiltration, which may be mediated by a reduction of ICAM‐1, IL‐1α and CINC‐1 expression.

Supported by NIH HL028982
Non‐thrombotic and haemocompatible amine‐modified graphene is a safer alternative to graphene oxide for biomedical use
Authors: Kulkarni Paresh Prahlad , Singh Sunil K , Singh Manoj K , Sonkar Vijay K , Grácio José J. A. , Dash Debabrata
Published: June 2021

Graphene, a two‐dimensional nanocarbon allotrope has grabbed the attention of scientists and has been explored for a wide range of biomedical applications. However, we have recently reported graphene oxide (GO) to be highly thrombogenic in mice by evoking aggregation of platelets the central players in haemostasis and thrombosis. This potentially limits biomedical applications of graphene. Surface chemistry of nanomaterials is a critical determinant of biocompatibility and thus differentially functionalized nanomaterials could exhibit varied cellular toxicity. We, therefore, evaluated the effect of amine‐modification of graphene on its platelet reactivity. Remarkably, our results revealed that unlike GO amine‐modified graphene (G‐NH2) neither stimulated human platelets in vitro, nor induce pulmonary thromboembolism in vivo in mice. Furthermore, it did not evoke hemolysis and exhibited no cytotoxicity towards THP‐1 cell line even on prolonged exposure. These findings contrasted strikingly with those from GO and RGO, which were found to exhibit significant platelet stimulatory and hemolytic effect. We conclude that, G‐NH2 is potentially safer for in vivo biomedical applications than other commonly investigated graphene derivatives. The work was funded by DBT, ICMR and DST, Government of India, and FCT, Portugal.
Enhancement of Autophagy by Simvastatin through Inhibition of Rac1‐mTOR Signaling Pathway
Authors: Wei Yu-Miao , Zhang Yang , Li Xiao-Xue , Xu Ming , Li Pin-Lan
Published: June 2021

In addition to their action lowering lower blood cholesterol levels, the statins have been found to modulate biological characteristics and function nof vascular smooth muscle cells (VSMCs) such as viability, proliferation, apoptosis, survival and contraction. The present study was designed to test whether simvastatin as a prototype enhances autophagy in VSMCs and thereby exerts their beneficial effects in atherosclerosis. Using Laser flow cytometry with a Cyto‐ID Green dye that detects autophagosomes, we found that simvastatin significantly increased autophagosome formation in VSMCs isolated from mouse coronary arteries. Western blot analysis also demonstrated that simvastatin significantly increased the protein expression of typical autophagy markers including LC3B and beclin‐1 in these mouse VSMCs. By activated Rac1‐GTPase pull‐down assay, simvastatin was shown to decrease basal Rac1‐GTPase activity and blocked angiotensin II‐induced Rac1 activation in VSMCs. Analysis of mTOR activity by assay of phosphorlylation of its substrate S6K demonstrated that simvastatin inhibited both basal and angiotensin II‐induced mTOR activity in VSMCs. Stimulation of mTOR activity by phosphatidic acid, an mTOR agonist, inhibited simvastatin‐induced autophagy in VSMCs. Moreover, simvastatin increased protein expression of a contractile phenotype marker calponin in VSMCs, while such increase in calponin expression was blocked by the autophagy inhibitor 3‐MA. Collectively, our results indicate that simvastatin inhibits Rac1‐mTOR pathway and thereby increases the autophagy in coronary VSMCs that may stabilize VSMCs in the contractile phenotype and prevent these cells from proliferation and growth (Supported by NIH grants HL‐091464, HL57244 and HL‐75316).
Membrane Stretch‐Induced Activation of TRPM4 in Cerebral Artery Smooth Muscle Cells
Authors: Yang Ying , Gonzales Albert L , Sanders Lindsey , Earley Scott
Published: June 2021

In cerebral artery smooth muscle cells (SMCs), the melastatin transient receptor potential (TRP) channel TRPM4 is a crucial mediator of pressure‐induced membrane depolarization, and is activated by Ca2+ released from inositol trisphosphate (IP3) receptors located on the sarcoplasmic reticulum. In the current study, we examined the mechanosensitivity of TRPM4 channels in native SMCs. TRPM4 channel activity was recorded under perforated patch clamp conditions as the plasma membrane was stretched by applying suction to the patch pipette. Increases in cation current proportional to the magnitude of applied suction were recorded. Mechanosensitive currents were inhibited by the selective TRPM4 blocker 9‐phenenthral but were unaffected by the BKCa channel blocker paxilline. Phospholipase C (PLC) cleaves the membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) into IP3 and diacylglycerol (DAG). Prior studies suggest that mechanical stress can stimulate PLC activity. Therefore, we tested the hypothesis that PLC activity is necessary for plasma membrane stretch‐induced activation of TRPM4 in cerebral artery myocytes. We find that the PLC blocker U73122 inhibits stretch‐induced TRPM4 activation, suggesting that PLC plays an important role in activation of TRPM4 by mechanical stimulus in cerebral artery smooth muscle cells. RO1HL091905(SE); F31HL094145‐01(AG).
Soluble epoxide hydrolase deficiency alters pancreatic islet size and improves glucose homeostasis in a model of insulin resistance.
Authors: LURIA AYALA R , Bettaieb Ahmed , Xi Yannan , Inoue Hiromi , Tsai Hsing-Ju , Imig John D , Haj Fawaz G , Hammock Bruce D
Published: June 2021

Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease. Increasing endogenous levels of epoxyeicosatrienoic acids (EETs) are known for their analgesic, antihypertensive, and antiinflammatory effects. The availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH), which metabolizes EETs to their less active diols. In this study, we tested the hypothesis that EETs are involved in glucose regulation and in retarding the development of insulin resistance. To address the role of EETs in regulating glucose homeostasis and insulin signaling, we used mice with targeted gene deletion of sEH and a subsequent study with a selective sEH inhibitor. When WT mice are fed a high fat diet, insulin resistance develops. However, KO or inhibition of sEH activity resulted in a significant decrease in plasma glucose. These findings are characterized by enhancement of tyrosyl phosphorylation of the insulin receptorand its downstream cascade. In addition, pancreatic islets were larger when sEH was disrupted. This effect was associated with an increase in vasculature. These data suggest that an increase in EETs due to sEH‐gene knockout leads to an increase in the size of islets and improved insulin signaling and sensitivity.
Increased MMP‐9 Activity During the Progression of Renal Injury in Type‐2 Diabetic Nephropathy rats
Authors: Williams Jan Michael , Slaughter Tiffani Nicole , Wells Adrienne , Rucker Carlos , Spires Denisha , Kyle Patrick B. , Roman Richard J.
Published: June 2021

We have reported that treatment with a matrix metalloproteinase (MMP) inhibitor, XL784, markedly prevents the progression of renal injury in type‐2 diabetic nephropathy (T2DN) rats. However, the MMP isoform(s) involved in the development of renal injury remains to be determined. The current study examined changes in the expression of MMP‐2 and ‐9 during the development of renal disease in T2DN rats that are susceptible to renal injury versus Goto‐Kakizaki (GK) diabetic rats that are nearly genetically identical but resistant to renal disease. GK and T2DN rats were uni‐nephrectomized at 6 months of age and followed until 9 months of age. At 9 months of age, MAP (measured by telemetry) was similar in GK and T2DN rats. Protein excretion increased to 263±54 mg/day in T2DN rats versus 43±6 mg/day in the GK strain. The kidneys of T2DN rats exhibited mesangial expansion, glomerulosclerosis, and interstitial and renal fibrosis. MMP‐9, but not MMP‐2, activity increased to a greater extent in T2DN rats in comparison to GK rats in the renal cortex during the development of renal disease. These data indicate that elevations in MMP‐9 activity may contribute to the progression of renal injury in T2DN rats and suggest that MMP inhibitors may be useful to prevent the progression of renal disease in diabetic patients. The current study was supported by a PHRMA Starter grant and NIH grants HL36279 and HL29587.
Cellular Localization of P0 (Adenine) Receptor in Rat Kidney
Authors: Kishore Bellamkonda K , Zhang Yue , Pop Ioana L , Gevorgyan Haykanush , Müller Christa E , Peti-Peterdi Janos
Published: June 2021

P0 receptor (R) is a Gi‐coupled receptor which potentially reduces cellular cAMP levels. It binds adenine with high affinity as compared to adenosine, AMP, ADP or ATP. Hence, we localized it in rat kidney. Real‐time RT‐PCR showed highest expression of P0‐R mRNA in the superficial cortex (CTX) followed by inner and outer stripes of outer medulla (OM), deep CTX, and the base of inner medulla (IM). Immunoblots using a peptide‐derived and affinity‐purified rabbit polyclonal antibody specific for an 18‐amino acid C‐terminal sequence of rat P0‐R which we generated, detected two bands between ~36–40 kDa (MW of native protein 37 kDa) in CTX, OM and IM, which were ablated by pre‐adsorption of the antibody with the immunizing peptide. Immunoperoxidase labeling showed strongest labeling in small followed by medium blood vessels. Strong labeling is also seen in the afferent arterioles. Diffuse and weaker labeling is seen on the brush border of proximal tubules and on the medullary thick ascending limbs. Sparse labeling is seen over the peritubular interstitial cells in the CTX. Both collecting ducts (CD) and connecting tubules showed labeling, with the latter having slightly stronger intensity. In the CD the labeling is mostly on the apical aspect of the principal cells. Thus, P0‐R is expressed in vascular and tubular elements of rat kidney, and thus it may play functional roles in renal hemodynamics and transport. Funding: VA & NKF
Enhanced renin release and content in juxtaglomerular (JG) cells from VAMP8 knockout mice
Authors: Mendez Mariela
Published: June 2021

Renin is essential for angiotensin II production and blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells. We showed that vesicle associated membrane protein 2 (VAMP2), a member of SNARE family, mediates stimulated renin exocytosis in JG cells. However, the VAMP isoform required for constitutive renin release is unknown. In other cells VAMP8 mediates granule maturation and constitutive exocytosis. We hypothesized that VAMP8 mediates constitutive renin release in JG cells. We used primary cultures of JG cells from wild type (WT) and VAMP8 knockout (V8‐KO) mice and measured VAMP8 localization and renin released to the medium during 1 hr. In wild type, VAMP8 was expressed in JG cell granules. Baseline renin release was 5‐fold higher in V8‐KO than WT (WT=0.06±0.01; KO=0.3±0.05 μgAngI/hr/mg prot.; p<0.05). Total renin content was 5‐fold higher in V8‐KO than WT (WT=2.9±0.2; KO=16.7±0.6 μgAngI/hr/mg prot.; p<0.05). The fraction of total renin released to the media was similar in V8‐KO and WT JG cells (WT: 1.6±0.3; KO: 1.7±0.5% of total renin) indicating higher number of granules in V8‐KO, or higher renin content/granule. Immunolabeling of renin and confocal imaging showed enlarged granules and vacuoles in JG cells from V8‐KO. We concluded that: 1) VAMP8 does not mediate constitutive renin exocytosis; and 2) VAMP8 may play an inhibitory role in transcription, translation or processing of renin. 5F32HL096346
Endothelium differentiation and regeneration from human adipose‐derived stem cell by shear stress
Authors: Chang Ya-Ju , Lin Sheng-Che , Chien Shu , Wu Chia-Ching
Published: June 2021

In the vascular system, endothelial cells (ECs) provide an interface between blood streams and vessel wall. The shear stress generating by blood flow might be a role to regulate endothelium differentiation and vascular development. Adipose‐derived stem cells (ASCs), isolated from adipose tissue, would be applied to regenerative medicine or cell‐based therapy. Thus, we want to investigate that endothelium differentiation of ASCs combines both biochemical and mechanical stimulation. Human adipose‐derived stem cells (hASCs) were isolated and cultured in DMEM, containing 10% FBS, and penicillin/streptomycin at 37¢J in 5% CO2. We combined the endothelial growth factors (EGM2) and shear stress system (12 dyn/cm2) to stimuli hASCs differentiation. RNA and protein level of EC markers were measured by RT‐PCR and western blotting. DiI‐LDL uptake and Matrigel assay were performed for EC functional analysis. Up‐regulation of EC marker and function reveal the synergistic effect of endothelium differentiation on hASCs under biochemaical and mechanical stimulation. This study was supported by NSC in Taiwan (99‐2320‐B‐006‐002‐MY3).
Active transepithelial Cl−‐secretion promotes hydrostatic lung edema
Authors: Solymosi Esther Andrea , Kaestle Stephanie M , Vadász István , Wang Liming , Morty Rory , Kuebler Wolfgang M
Published: June 2021

Hydrostatic lung edema evolves from increased fluid filtration and inhibition of epithelial Na+ channels (ENaC) that facilitate alveolar fluid clearance. Recently, we identified alveolar fluid secretion (AFS) as new key component in edema formation. Here, we tested whether Cl− secretion via cystic fibrosis transmemberane conductance regulator (CFTR) and Na+‐K+‐Cl− cotransporter 1 (NKCC1) may mediate AFS at elevated left atrial pressure (PLA) and may be induced by inhibition of ENaC.

In isolated lungs, we quantified AFS by a double indicator dilution technique, and transepithelial Cl− flux by radionuclide tracing and alveolar Cl− imaging. PLA elevation induced lung edema and AFS that coincided with transepithelial Cl− secretion and alveolar Cl− influx. These effects were blocked by inhibitors of CFTR, NKCC or Na+‐K+‐ATPase, and CFTR−/− mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride at physiological

PLA induced AFS and Cl− secretion that were again CFTR‐, NKCC‐ and Na+‐K+‐ ATPase dependent.

We conclude that transepithelial Cl− and fluid flux reverse from absorptive to secretory mode at hydrostatic stress as a result of ENaC inhibition, and are mediated by NKCC and CFTR.
Metabolomics Reveals the role of hypoxia‐inducible factors in hypoxia‐induced liver metabolic shift
Authors: Qu Aijuan , Li Fei , Krausz Kristopher , Shah Yatrik M. , Gonzalez Frank J.
Published: June 2021

Hypoxia‐inducible factor (HIF) is critical in the cellular adaptation to low cellular oxygen. A sustained increase in HIF signaling leads to spontaneous steatosis, inflammation and liver tumorigenesis. However, the metabolic responses and signaling networks regulated by HIFs in the liver are unclear. In the current study, serum from liver‐specific Von Hippel‐Lindau (Vhl)‐deficient mice overexpressing HIF was analyzed by metabolomics using LC‐MS. A dramatic decrease in metabolites involved in fatty acid β‐oxidation, which was correlated with the suppression of genes responsible for β‐oxidation, indicating a mitochondrial fatty acid oxidation defect. GC‐MS data revealed a sustained enrichment of lactate and alanine in serum of Vhl‐deficient mice. However, glutamine, an important energy substrate was robustly increased with these mice. These metabolomics data demonstrate that HIF is responsible for a metabolic shift from oxidative phosphorylation to glycolysis under hypoxia. In addition, many bile acids were found dramatically accumulated in Vhl‐deficient mice, indicating a disruption of liver bile acid metabolism. Taken together, these data reveal a central role for HIF in hepatic and systemic metabolic reprogramming under hypoxia and provide potential biomarkers for liver diseases. This study was funded by National Cancer Institute Intramural Program.
Inhibition of hypoxia inducible factor 1 alpha (HIF‐1a) improves the metabolic phenotype in C57BL/6J mice with diet induced obesity (DIO)
Authors: Shin Mi-Kyung , Drager Luciano , Bevans-Fonti Shannon , Yoo Doo Young , Aja Susan , Bhanot Sanjay , polotsky Vsevolod
Published: June 2021

Obesity has been associated with tissue hypoxia. Hypoxia inducible factor 1 (HIF‐1) is a master‐regulator of metabolic responses to hypoxia, but its role in obesity is unknown. DIO mice were treated with antisense oligonucleotides (ASO) against an O2‐ sensitive α subunit of HIF‐1 or control ASO (25 mg/kg, i.p., twice a week) for 8 weeks and compared with an unhandled group. HIF‐ 1α ASO suppressed HIF‐1α gene expression by 95% in liver and epididymal fat and by 72% in brown fat, whereas inguinal, mesenteric fat and muscle were unaffected. By Day 28th, HIF‐1a ASO mice started losing weight and by the end of the experiment they were 10% lighter then mice in the control ASO and unhandled groups (41 ± 3.9 vs 46 ± 3.1 and 47 ± 2.8 g respectively, p < 0.001). There was no change in food intake, but there was a significant increase in the energy expenditure (13.3 ± 0.6 vs 12 ± 0.1 and 11.9 ± 0.4 kcal/kg/hr in control ASO and unhandled groups, p < 0.001) and activity. HIF‐1α ASO mice showed a significant decreases in fasting blood glucose (195.5 ± 8.4 mg/dl vs 239 ± 7.8 mg/dl and 222 ± 8.2 mg/dl, p < 0.01), fasting plasma insulin (0.29 ± 0.04 vs 0.53± 0.09 and 0.77 ± 0.18 ng/ml, p < 0.05), liver fat content (15.32 ± 1.7 μg/mg vs 23.07 ± 1.5 μg/mg and 22.87 ± 21.1 μg/mg, p < 0.05), and increased liver glycogen content (12.1 ± 0.8 % wt/wt vs 8.9 ± 0.9, p < 0.05, and 7.4 ± 1.2 % wt/wt, p < 0.01), compared to control ASO and unhandled mice, respectively. In conclusion, in DIO mice, depletion of HIF‐1α results in increased energy expenditure, weight loss, improved insulin resistance and glucose tolerance.
The DNA Glycosylase Ogg1 is Required for Hypoxia‐induced Activation of VEGF Transcription in Pulmonary Artery Endothelial Cells (PAECs)
Authors: Pastukh Viktor M. , Bardwell Gina C. , Patel Mita , Al-Mehdi Abu-Bakr , Gillespie Mark N.
Published: June 2021

Hypoxia causes an oxidant stress accompanied by transient oxidative base modifications in hypoxic response elements (HREs) in promoters of inducible nuclear genes. Time‐dependent elimination of the base modifications suggests that they are removed by the base excision pathway of DNA repair (BER), but how DNA repair interacts with transcription is unknown. We tested the idea that suppression of BER by siRNA‐mediated knockdown of the first enzyme in the sequence, Ogg1, altered transcriptional complex formation on the VEGF HRE and attenuated VEGF mRNA expression. Hypoxia increased the number of HRE sequences harboring 8‐oxoG and associating with Hif‐1, as determined by ChIP analysis. While knock‐down of Ogg1 increased the proportion of sequences containing 8‐oxoG, Ogg1 deficiency attenuated Hif‐1 incorporation into the transcriptional complex and reduced VEGF mRNA expression. Knock‐down of Ogg1 expression failed to impact hypoxia‐induced oxidant generation in PAECs nor did it alter the rise in nuclear Hif‐1 or its ability to bind to an oligonucleotide model of the VEGF HRE. These findings indicate that hypoxia causes formation of 8‐oxoG in the VEGF of HRE and suggest that the BER pathway, recruited in response to the hypoxia‐induced oxidative base modifications, may be important for transcriptional activation. Sponsored by NIH.
Novel role for copper transport protein CTR1 in hypoxia induced pulmonary hypertension
Authors: Zimnicka Adriana Monika , Chen Jiwang , Yuan Jason X.-J.
Published: June 2021

Pulmonary hypertension (PH) is a serious disorder that leads to right heart failure. It is characterized by elevated pulmonary vascular resistance and high pulmonary arterial pressure due to sustained vasoconstriction and remodeling of the vascular wall. It is known that cellular copper (Cu) plays an important role in angiogenesis, remodeling of extracellular matrix, and defense against oxidative radical damage, but no study has related abnormal changes in Cu‐transport to the pathogenesis of PH. Preliminary studies from our lab indicate that in a chronic hypoxia mouse model of PH both, the Cu import protein CTR1 and the Cu export protein ATP7A, are up‐regulated in lung, while Cu,Zn‐SOD, involved in neutralizing superoxide radicals, is down‐regulated. Furthermore, hypoxia results in up‐regulation of LOX (Cu‐dependent lysyl oxidase) that plays an important role in crosslinking extracellular matrix collagen and elastin fibers. Similar observations are made in sub‐cultured pulmonary artery smooth muscle cells (PASMC) after 48 hours of hypoxia, or in the presence of cobalt, a hypoxia inducer. Our observations indicate that cellular Cu metabolism is sensitive to hypoxia through transcriptional regulation of CTR1 and may contribute to the development of pulmonary hypertension through increased reactive oxygen species (ROS) and LOX‐dependent vascular remodeling of adventitia.
Arterial pressure and ventilation in sodium depleted rats
Authors: De Paula Patricia M , Favero Michele T , Menani Jose Vanderlei
Published: June 2021

In the present study, we investigated the cardiorespiratory responses to sodium depletion (SD) in awake rats. Male Holtzman rats (300 g, n=9) with catheters inserted into the abdominal aorta, for recording mean arterial pressure (MAP) and heart rate (HR), were used. Ventilation (VE) was measured by whole body plethysmograph method. MAP, HR and VE were recorded in normovolemic (NORMO, before SD), in depleted (24 h of SD) and repleted rats (after two hours of access to 1.8% NaCl and water). SD was produced by the treatment with the diuretic furosemide (20 mg/kg of body weight) injected subcutaneously followed by 24 h of sodium‐deficient diet. SD did not modify baseline MAP (104±4 mmHg, vs. NORMO: 105±4 mmHg) or baseline HR (334±20 bpm, vs. NORMO: 379±13 bpm), however increased VE (708±107 ml/min/kg, vs. NORMO: 478 ± 60 ml/min/kg, p<0.05). The increase in VE was due to the increase in the tidal volume (VT, 7±0.6 ml/kg, vs. NORMO: 5±0.4 ml/kg, p<0.05), without changing respiratory frequency (fR, 99±8 cpm vs. NORMO: 85 ± 6 cpm). An increased VE similar to SD was maintained in repleted rats (640±33 ml/min/kg). The results suggest that sodium depletion may affect the modulation of baseline VT producing increases in VE, without significantly changing fR.

Supported by: CAPES, FAPESP and CNPq
Effect of acetylcholinesterase blockade with pyridostigmine on baroreflex and cardiovascular autonomic control in heart failure rats, six to seven weeks after coronary artery ligation
Authors: Sabino Joao Paulo Jacob , da Silva Carlos Alberto Aguiar , Junior Rubens Fazan , Salgado Helio Cesar
Published: June 2021

We evaluated in conscious heart failure (HF) rats the effect of acetylcholinesterase blockade with pyridostigmine (PYR) on systolic arterial pressure (SAP) variability (spectral analysis), baroreflex effectiveness index (Δ heart rate/Δ mean arterial pressure; bpm/mmHg), parasympathetic control of the heart (Δ heart rate after atropine; bpm) and intrinsic heart rate (heart rate after atropine plus propranolol; bpm). HF rats showed smaller power in the low frequency (LF, mmHg2) band of SAP spectrum [1.2±0.2 (n=10) vs. 3.3±0.4 in control rats (n=9)] while PYR prevented this reduction [2.9±0.8 (n=9)]. The baroreflex effectiveness index was attenuated in HF rats [−1.3±0.1 (n=9) vs. − 2.4±0.4 in control rats (n=11)] while PYR prevented this attenuation [−2.4±0.3 (n=9)]. The parasympathetic tonus was attenuated in HF rats [39±12 (n=7) vs. 90±15 in control rats (n=7)] while PYR prevented this attenuation [77±7, (n=6)]. Intrinsic heart rate was reduced in HF rats [363±7 (n=13) vs. 401±8 in control rats (n=14)], while PYR impaired this reduction [408±5 (n=12)]. In conclusion, blockade of acetylcholinesterase with PYR in HF rats was efficient to protect the autonomic control of SAP variability, the parasympathetic tone to the heart, the baroreflex sensitivity and the intrinsic heart rate. Therefore, an increase in parasympathetic function plays a benefic role in the development of HF.
Effect of pyridostigmine on hemodynamics and arrhythmias acutely after myocardial infarction in anesthetized rats
Authors: DOS SANTOS FERNANDA MACHADO , da Silva Carlos Alberto Aguiar , Salgado Helio Cesar , Fazan Rubens
Published: June 2021

We evaluated the effect of acute increase in parasympathetic function, elicited by pyridostigmine (PYR), shortly after myocardial infarction (MI) in anesthetized rats. Acute MI was elicited in urethane anesthetized rats by left coronary artery ligation under continuous recording of electrocardiogram (ECG) and arterial pressure (AP). Ten min after MI the rats were treated with PYR (0.25 mg/kg, iv, N=10) or saline (control, N=8) and ECG/AP recordings lasted 4 hours. At the end of experiments, the hearts were removed and perfused with Evans’ blue to evaluate the infarcted area. MI size was similar between PYR treated (52±4%) and control rats (46±4%). A fall in mean AP (115±3 to 83±5 mmHg) and a trend to bradycardia (450±6 to 436±8 bpm, p=0,051) were observed immediately after MI. AP remained 46 to 54 mmHg lower than basal values during the 4 hours after MI and PYR did not affect this parameter. A tachycardia was observed 3 and 4 hours after MI (38±17 and 45±15 bpm, respectively) in control rats. PYR elicited a prompt bradycardia (−94±19 bpm) and prevented the tachycardia 3 and 4 hours after MI. Premature ventricular beats (PVB) were more frequent in control as compared to PYR treated rats. Segments of 2,000 beats were examined every hour after MI to identify PVB. Four out of 8 control rats showed 1 to 15 PVB while only 2 out of 10 PYR treated rats showed 1 to 4 PVB within the 4 h after MI.
Microinjection of kynurenic acid (KA) into the medial reticular formation elicits behavior‐dependent effects on cough and swallow motor patterns in the anesthetized cat
Authors: Pitts Teresa , Poliacek Ivan , Rose Melanie J , Mortensen Ashley N , Davenport Paul W , Morris Kendall F , Bolser Donald C
Published: June 2021

Kynurenic acid (KA) affects multiple receptors and is elevated in the brainstem during neurological diseases, such as Alzheimer's and Parkinson's. Aspiration pneumonia associated with dysphagia and dystussia is common in these disorders. The medial reticular formation (MRF) caudal to obex is important for cough coordination. We speculated this region is also important for swallow. Electromyograms of geniohyoid (GH), thyroarytenoid (ThAr), upper esophageal sphincter (UES), thyrohyoid (TH), parasternal (PS) and transversus abdominis (TA) were recorded in anesthetized cats. Cough and swallow were elicited by tracheobronchial stimulation and intraoral water injection respectively. KA (50nL; 50mM) was microinjected bilaterally at 6 locations within the MRF. During swallow, KA induced differential effects on cranial motor activity (decreased: TH, ThAr, and GeHy and increased: UES), but the spinal motor component (PS) did not change. During cough, cranial motor drive did not change, but there was a decrease in motor drive to spinal motoneuron pools (PS, TA). The results support multiple effects of KA including direct actions at/near the injections and secondary synaptic effects on anatomically distant neural assemblies. The data support the existence of common neuronal assemblies in the MRF for swallow and cough. Support: HL 89104; HL 103415.
GAL‐021 acts as a novel respiratory stimulant in non‐human primates
Authors: Golder Francis John , Wardle Robert L , Van Scott Michael R , Hoskins Paul A , Dax Scott L , Peng Sean , MacIntyre D Euan , Mannion James C
Published: June 2021

Opioid‐induced respiratory depression (OIRD) is a common side effect of opioid‐induced analgesia and can be life‐threatening, especially during the perioperative period and sleep. Current therapy for OIRD includes administering opioid antagonists and/or decreasing subsequent opioid doses, both of which can compromise analgesia. We have demonstrated that GAL‐021, a novel respiratory stimulant, increases minute ventilation (Vmin) and reverses OIRD in rats, without diminishing opioid‐induced analgesia. Here, we describe the effects of GAL‐021 in Cynomolgus monkeys. In isoflurane‐anesthetized opioid‐naïve monkeys, GAL‐021 administered by IV bolus (0.01 – 0.3 mg/kg) or infusion (0.02 – 0.1 mg/kg/min) increased Vmin, tidal volume, and respiratory frequency. In conscious animals, OIRD was induced by morphine (2.4 – 3.2 mg/kg, slow IV bolus), titrated in each animal to decrease Vmin by 20 to 30% and increase ETCO2 by 10 mmHg. Administration of GAL‐021 15‐min post‐morphine diminished OIRD in a dose‐dependent manner. Loading GAL‐021 at 0.1 mg/kg/min × 5 min and maintaining infusion at 0.05 mg/kg/min × 10 min reduced OIRD by 75%. In contrast, loading at 0.2 mg/kg/min × 5 min and maintaining infusion at 0.1 mg/kg/min × 10 min returned ETCO2 to pre‐morphine levels. These data demonstrate that GAL‐021 stimulates respiration in conscious and anesthetized monkeys and is capable of reversing OIRD in non‐human primates.
Neuronal pentraxin 1: a molecular determinant of hypoxic‐ischemic brain injury
Authors: Hossain Mir Ahamed
Published: June 2021

The mechanism(s) involved in neonatal hypoxic‐ischemic brain injury remain poorly understood. Here, we report the induction of a novel neuronal protein ‘neuronal pentraxin 1’ (NP1) in brain injury in neonatal animal model of hypoxia‐ischemia (HI). Neonatal brains developed marked infarcts at 24 h and showed significant loss of ipsilateral cortical and hippocampal volumes at 7 d post‐HI. Elevated expression of NP1 was observed in cortical layers and in the hippocampal CA1 and CA3 areas following HI; same brain areas that developed infarcts and increased TUNEL‐positive cells. In contrast, NP1 knockout (NP1‐KO) and NP triple KO (NP‐TKO) neonatal mice showed complete absence of brain infarction following HI compared to the wild‐type (WT) animals; supporting a role for NP1 in brain injury. Primary cortical and hippocampal neurons showed an OGD time‐dependent induction of NP1 and subsequent increase in cell death; consistent with in vivo findings. NP1 gene silencing in WT cortical neurons by NP1‐siRNA significantly reduced neuronal death, and NP1 (−/−) null cortical neurons were significantly protected against OGD. Overexpression of NP1 by infecting WT and NP1‐KO cells with pLenti6v5‐Nptx1 further enhanced the OGD‐induced neuronal death. Our findings point to a novel molecular target of brain injury, and will foster new strategies for clinical management of neonates suffering from HI. Supported by RO1NS046030.
A rare pattern of brachial artery variation
Authors: Jacomo Alfredo Luiz , Akamatsu Flávia , Saleh Samir , Andrade Mauro
Published: June 2021

Introduction

Unlike venous system, variations in arterial anatomy are less frequent and most of them affect visceral arteries. In limbs, variations of the brachial artery are the most reported and at least six different patterns have been described so far. The commonest is the superficial brachial artery which lies superficially to the median nerve. Much less prevalent are the high origin of the radial artery (brachioradial artery) or the existence of a doubled brachial artery (accessory brachial artery). We present a previously undescribed pattern of brachial artery variation.

Description

During dissection of the right upper limb of a 60 year‐old male embalmed cadaver, we found the bifurcation of the brachial artery in the proximal portion of the middle third of the arm. Its medial branch reaches the medial aspect of the arm, posterior to the median nerve. Afterwards, this medial branch redirects laterally and crosses again the median nerve, this time lying anterior to the nerve until it reaches the lateral aspect of the arm. At the elbow level, the medial branch originates the radial artery. The lateral branch of the brachial artery remains lateral to the median nerve and continues as ulnar artery and originates the interosseus artery.
Validity and Reliability of a Novel 3D Measurement Approach of the Acetabulum
Authors: Martin Charys M , Turgeon James G , Rice Charles L , Wilson Timothy D
Published: June 2021

The hip joint is a frequent site of osteoarthritis. Advances in diagnosis and treatment are limited due to the lack of reliable measures for quantifying hip morphology. Current diagnostic measures of the hip are performed with predetermined measures in 2D planes, but these do not fully account for morphological variation nor do they utilize 3D capabilities of CT and MR. A valid and reliable measurement modality describing the entire geometry of the hip is necessary for early diagnosis and treatment of hip disease. A 3D measurement approach was developed and proved to be valid and reliable for the proximal femur. The purpose here was to assess the validity and reliability of the same 3D approach on acetabular morphometry. The technique was applied to 45 cadaveric acetabuli (23 Right; 9 Female) and their digitally reconstructed CT models. Preliminary results indicate this measurement approach is not valid with significant differences detected between the cadaveric and digital measurements, p < 0.05. However, the digital measurements had excellent intraobserver reliability (ICC = 0.99) and interobserver reliability (ICC = 0.94). Although this approach was valid and reliable for proximal femora it appears reliable, but not valid for measuring the acetabuli. This result is due to the presence of calcified labra being visualized as bone, and being included in the bone measurements, when measuring the digital models.

Grant Funding Source: Departmental Funding
Possible convergent evolution of yellow psittacofulvin colors in the feathers of cockatoos (Cacatuidae) and parrots (Psittacidae)
Authors: Bonin Jonathan , Homberger Dominique
Published: June 2021

Psittacofulvins (pigments unique to Psittaciformes) produce a yellow feather color that may fluoresce in some species (Voelker 1937). To test the hypothesis that these colors may have evolved independently in cockatoos and parrots, yellow feathers were analyzed spectrometrically in four Australasian cockatoos (Calyptorhynchus banksii, C. funereus, Cacatua galerita, and Nymphicus hollandicus) and three parrots (the Australian Melopsittacus undulatus, the Caribbean Amazona ventralis, and the South American Ara ararauna). The spectra of the studied species have a peak in the UV range, a reduced reflectance in the violet‐blue range, and the highest reflectance in the green‐to‐red range. In addition, the spectra of cockatoos show an increasing reflectance within the blue‐green range, whereas the spectra of Melopsittacus and Amazona show an increasing reflectance within the green range and the spectrum of Ara an increasing reflectance within the green‐yellow range. Only Calyptorhynchus and Nymphicus showed fluorescence. Hence, cockatoos are characterized not only by several morphological features, but also by the reflectance spectra of their yellow feathers, thereby further supporting the hypothesis that cockatoos evolved their psittaciform morphotype independently from parrots (see Homberger 2003). Funding: LSU Foundation to DGH.

Grant Funding Source : N/A