bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperglycemia up‐regulates AT2 receptors via IRF‐1 in proximal tubule epithelial cells.
Authors: Ali Quaisar , Hussain Tahir
Published: June 2021

We have reported that the renal AT2 receptor expression and their function on natriuresis are increased in hyperglycemic obese rats. However, role of hyperglycemia and the mechanism responsible for increase in AT2 receptor expression is not known. The present study was designed to test the hypothesis that hyperglycemia increases AT2 receptor expression by transcriptional activation via interferon regulatory factor 1 (IRF1) in the renal proximal tubules. Western blotting and qRT‐PCR studies revealed that AT2 and IRF1 protein/mRNA expression in the proximal tubule (PTs) of obese rats was greater than in lean rats. To study the cause‐effect relationship, we used HK‐2 cells. Twenty hr incubation of HK2 cells with high glucose concentration (25 mM) caused an increase in the AT2 receptor protein/mRNA and the IRF1 expression. To study the role of IRF1 in hyperglycemia‐induced AT2 expression, we knocked‐down IRF1 using IRF1 siRNA (100 nM). Treatment with siRNA decreased IRF1 as well as AT2 receptor expression and prevented high glucose‐induced increase in the expression of both the IRF1 and AT2 receptor. The data suggest that hyperglycemia induces a transcriptional up‐regulation of AT2 receptor expression via increasing the IRF1 expression. This mechanism may be compensatory to promote natriuresis and prevent blood pressure increase in obese rats, as shown earlier.
Differences in beta arrestin dependent signaling by PAR2 and NK1R are determined by the C‐terminal tails
Authors: Pal Kasturi , DeFea Kathryn
Published: June 2021

Protease Activated Receptor 2 (PAR2) is a G protein coupled receptor (GPCR). Activation takes place by tryptic cleavage of the N terminus releasing a tethered ligand, leading to signaling via Gαq pathway, generating Protein kinase C (PKC). The paradigm of GPCR signal attenuation, suggests that PKC and GPCR kinases(GRK) phosphorylate the C terminal of PAR2, recruiting β arrestins ½ to the receptor. This either leads to receptor desensitization or arrestin dependent extracellular signal regulated kinases 1 and 2 (ERK1/2) activity, promoting chemotaxis. Neurokinin 1 receptor (NK1R), another GPCR, on activation by Substance P activates similar signaling cascades. NK1R mediated ERK1/2 activation, leads to nuclear localization and cell division. NK1R and PAR2 differ in mode of endocytosis, intracellular sorting and mechanism of resensitization. We hypothesize that these differences in arrestin dependent signaling by the two receptors depend on their C termini. Bioluminescence resonance energy transfer (BRET) has shown that PAR2 recruits both β arrestin1 and 2. NK1R shows preferential recruitment of β‐arrestin 2. The rates of β‐arrestin recruitment to PAR2 are accelerated than NK1R. Using specific inhibitors of the members of the Gαq signaling cascade and PKC phosphorylation mutant of PAR2, we show that β‐arrestins ½ recruitment to PAR2 is independent of receptor phosphorylation and Ca2+ mobilization.
Identification of Novel Isoforms of Regulator of G‐Protein Signaling 6 (RGS6)
Authors: Yang Jianqi , Ibeawuchi Stella‐Rita , Huang Jie , Stewart Adele , Chen Ching‐Kang Jason , Fisher Rory A
Published: June 2021

RGS6 is a member of the RGS family of proteins that play critical roles in attenuating G protein‐coupled receptor signaling. In addition to a hallmark RGS domain that mediates inactivation of Gi/o subunits, certain RGS6 isoforms possess DEP and GGL domains implicated in their membrane anchoring and binding to Gβ5, respectively. Half of the 36 splice variants of RGS6 we identified in human brain encode DEP domain‐containing long isoforms of RGS6 (RGS6L). In this study, we generated a highly specific antibody against RGS6L isoforms. Using this antibody to evaluate expression of RGS6L in mouse tissues, we identified an immuno‐reactive band corresponding to the predicted molecular weight of RGS6L isoforms in numerous tissues including brain, cerebellum and spinal cord. Moreover, additional unique bands were present in brain, liver and kidney. The size of these other immuno‐reactive proteins could not be accounted for by any known RGS6L transcript. Both the predicted and unique RGS6L immuno‐reactive bands were present in lysates derived from wild‐type but not RGS6 knockout mice, suggesting that the unique bands represent novel isoforms of RGS6L. This is the first evidence that multiple and novel isoforms of RGS6L are present in various mouse tissues. The biological function of the novel isoforms of RGS6L are under investigation. (Supported by NIH GM075033, AHA 0750057Z).
Platelet P‐selectin mediates cytomegalovirus‐induced microvascular dysfunction
Authors: Stokes Karen , Leskov Igor L , Senchenkov Evgeny V
Published: June 2021

Cytomegalovirus (CMV) infects a majority of the population worldwide. It has been implicated in cardiovascular disease (CVD) possibly through its inflammatory actions. Although platelets and platelet‐associated P‐selectin (P‐sel) are linked to inflammatory responses in CVD, their role in CMV‐induced vascular responses is unknown. We assessed the role of platelets in CMV‐induced microvascular inflammation. Wildtype (WT) mice and bone marrow chimeric mice deficient in platelet P‐sel (P‐sel Ch) received mock or murine CMV (mCMV) IP. Mice were fed normal chow (ND) or placed on high cholesterol diet (HC) at 5wk p.i. to investigate the synergism between mCMV & HC. Microvascular responses were measured at 11wk. Some WT mice were depleted of platelets 24h before observation. mCMV−/+HC caused significant endothelial dysfunction in arterioles. Vasodilation in mCMV‐ND P‐sel Ch, but not platelet depleted mice, was restored to Mock‐ND levels. Partial protection was seen in mCMV+HC mice with platelet depletion or in P‐sel Ch. Only mCMV+HC elevated leukocyte recruitment in venules. This was reduced to mock levels by acute platelet depletion, but not in P‐sel Ch. Our findings implicate a novel role for platelets, acting through P‐sel, in CMV‐induced arteriolar dysfunction, and suggest that the addition of HC leads to a platelet‐dependent, P‐sel independent inflammatory infiltrate. (AHA‐0730294N & NIH‐P20RR018724)
Arsenic‐induced endothelial activation: contribution of endoplasmic reticulum stress
Authors: SRIVASTAVA SANJAY , SITHU SRINIVAS D , VLADYKOVSKAYA ELENA , HABERZETTL PETRA , D'SOUZA STANLEY E
Published: June 2021

Exposure to arsenic exacerbates atherosclerosis, however, mechanisms of atherogenic affects of arsenic are not known. In the present study, we show that exposure of endothelial cells (EC) to arsenic significantly (P<0.01) increased the expression of IL‐8, permeability of endothelial cell monolayer and transmigration of leukocytes. Arsenic also increased the expression of ER‐resident chaperones ‐ GRP78, GRP94 and HERP by 1.4–5.4‐fold. Moreover, arsenic, induced the phosphorylation of eIF2á, and the expression of its downstream transcription factors such as ATF3 and CHOP by 2–6‐fold. Exposure to arsenic also led to the splicing of the bZIP transcription factor, XBP‐1. Adenoviral transfection of EC with ATF‐6 increased the expression of several ER chaperones and inhibited the arsenic‐induced IL‐8 expression and transmigration of leukocytes by >50 %. Anti‐IL‐8 antibody and antagonist of IL‐8 receptor (CXCR‐2), SB225002, also significantly (P<0.01) inhibited the arsenic‐induced transmigration of leukocytes. These data suggest that arsenic‐induced cytokine production can increase endothelial activation and leukocytes transmigration in a ER‐stress dependent manner. ER‐stress therefore, could be an important regulator of endothelial activation and atherogenesis.
MLCK210 deficiency ameliorates atherosclerosis in apoE knockout mice
Authors: Sun Chongxiu , Wu Mack H. , Yuan Sarah Y.
Published: June 2021

Endothelial barrier dysfunction is a key event in the pathogenesis of atherosclerosis. The objective of this study was to explore the role and mechanisms of MLCK210 (non‐muscle myosin light‐chain kinase) in the development of atherosclerosis in mice with an apoE knockout background. Histopathological examination demonstrated that MLCK210 deficiency reduced atherosclerotic lesion and macrophage deposit. Western blot and RT‐PCR analyses revealed MLCK210 expression in the aortic endothelium and peripheral monocytes. By measuring transendothelial electric resistance, we found that MLCK210 deficiency attenuated aorta endothelial hyperpermeability to pro‐inflammatory stimuli, including thrombin, ox‐LDL and TNF‐alpha. In monocytes, MLCK210 deficiency reduced their transendothelial migration in response to murine MCP‐1. Further mechanistic studies showed that these inflammatory mediators induced Src phosphorylation in endothelial cells and monocytes. The phosphorylation response was attenuated in MLCK210 deficient cells, corresponding to reduced endothelial permeability and monocytes transmigration. Taken together, the data suggest that MLCK210 contributes to the development of atherosclerosis by regulating endothelial barrier function and monocyte migration via a mechanism involving Src signaling. Supported by NIH HL61507, HL84542, HL70752, and HL73324.
PAR4 Deficiency Inhibits Microvascular Inflammation after Cerebral Ischemia/Reperfusion Injury
Authors: Mao Yingying , Zhang Ming , Kunapuli Satya , Tuma Ronald
Published: June 2021

Stroke is the second leading cause of death worldwide and antithrombotic therapy targeting thrombin signaling is one of the new treatments for ischemic stroke. Protease‐activated receptor 4 (PAR4) is the major thrombin receptors expressed in murine platelets. The role of PAR4 in cerebral ischemia/reperfusion injury was investigated by using a mouse transient middle cerebral artery occlusion/reperfusion (MCAO/R) model. Platelet/endothelial and leukocyte/endothelial interactions, which have been shown to play a critical role in the inflammatory responses during cerebral ischemic/reperfusion injury, were studied via a cranial window and intravital microscopy. We demonstrated that PAR4 −/− mice had a significant reduction in infarct volume at both 24 hours at 48 hours (p<0.001) following 1 hour MCAO compare to wild‐type mice. Furthermore, deficiency of PAR4 significantly decreased both platelet/endothelial and leukocyte/endothelial interactions. In addition, we also found that blood‐brain barrier (BBB) disruption and edema formation were dramatically attenuated in PAR4−/− mice. In conclusion, PAR4 deficiency protects against cerebral ischemic injury partially though attenuation of cerebral microvascular inflammation. Therefore, PAR4 may represent therapeutic target for the treatment ischemia injury.
Activation of calcium‐independent phospholipase A2β increases platelet activating factor and PGI2 production in cardiac endothelial cells
Authors: Sharma Janhavi S , McHowat Jane
Published: June 2021

The endothelium is a critical barrier between the circulation and underlying tissues. We have previously shown that activation of protease‐activated receptor‐1 (PAR‐1) and PAR‐2 on the surface of endothelial cells (EC) by tryptase or thrombin increases calcium‐independent phospholipase A2β (iPLA2β) activity resulting in the production of multiple membrane phospholipid‐derived inflammatory metabolites. We isolated EC from hearts of iPLA2β knockout (KO) and wild type (WT) mice and measured arachidonic acid (AA), prostacyclin (PGI2) and platelet‐activating factor (PAF) production in response to PAR stimulation. Thrombin stimulation (0.1 IU/ml) of WT EC rapidly increased AA (0.89 + 0.07 to 2.23+0.13%) and PGI2 release (75.0 + 2.8 to 133.7 + 3.4 pg/ml) and increased PAF production (1216 + 79 to 2308 + 259 dpm). Similar increases were observed with tryptase (20 ng/ml) stimulation. Thrombin and tryptase‐stimulated responses were inhibited by pretreatment of WT EC with S‐bromoenol lactone ((S)‐BEL, 5μM, 10mins), which selectively inhibits iPLA2β. Thrombin or tryptase stimulation of iPLA2β KO EC demonstrated no significant increase in PAF production and inhibited AA and PGI2 release. These results indicate that iPLA2β is the predominant isoform in EC and could contribute to inflammation via PGI2 and PAF production.
Cystathionine β‐synthase and cystathionine γ‐lyase double gene transfer ameliorated homocysteine‐mediated mesangial inflammation through hydrogen sulfide generation
Authors: Sen Utpal , Qipshidze Natia , Abe Oluwasegun , Metreveli Naira , Tyagi Suresh
Published: June 2021

In the present study we investigated the in vitro signaling mechanism of Hcy‐mediated inflammation and determined the regulatory role of hydrogen sulfide (H2S) in this process. Mesangial cells were incubated with Hcy (75 μM) and supplemented without or with H2S (50 μM) for 48h. Inflammatory molecules MCP‐1 and MIP‐2 were measured by ELISA in the cultured supernatant. In the Hcy treated cells, H2S was measured in the supernatant. Hcy‐mediated phosphorylation of JNK and ERK were measured by Western blot. To enhance the endogenous production of H2S and clearance of Hcy, cystathionine β‐synthase (CBS) and cystathionine γ‐lyase (CSE) genes were delivered to the cells. Our results showed that Hcy upregulated inflammatory molecules MCP‐1 and MIP‐2; whereas the endogenous production of H2S was attenuated. H2S treatment as well as CBS and CSE double gene transfer markedly reduced Hcy‐induced upregulation of MCP‐1 and MIP‐2 in the culture supernatant. Hcy phosphorylated JNK and ERK, and H2S supplementation as well as CBS and CSE double gene transfection attenuated Hcy‐induced phosphorylation of these two pathways. Similarly, the inhibition of JNK and ERK phosphorylation by pharmacological and siRNA blockers attenuated MCP‐1 and MIP‐2 expressions induced by Hcy. We conclude that H2S plays a regulatory role in Hcy‐induced inflammatory mechanism and that JNK and ERK are the two signaling pathways involved in this process.
Acute hyperglycemia augments neurovascular injury in diabetes
Authors: Ergul Adviye , Elgebaly Mostafa M , Ogbi Safia , Johnson Maribeth , Mezzetti Erin , Fagan Susan C
Published: June 2021

Background

Admission hyperglycemia impacts ischemic stroke deleteriously but the relative role of acute hyperglycemia versus diabetes in the pathogenesis of this poor outcome is not clear.

Purpose

To test the hypothesis that the impact of acute hyperglycemia on neurovascular damage following ischemic injury is more severe when superimposed on diabetes.

Methods

Acute hyperglycemia (HG) was achieved by 3 ml IP glucose injection (300 mM) 20–30 minutes before 3 h middle cerebral artery occlusion (MCAO) in male control Wistar and mildly diabetic Goto‐Kakizaki (GK) rats. The goal was to achieve mild elevation in blood glucose levels (~180 mg/dl) in control rats to mimic glucose levels normally observed in the GK model. Following 21 h reperfusion neurobehavioral tests were performed before sacrifice and neuronal (infarct size) and vascular (hemorrhagic transformation (HT) frequency and excess hemoglobin) damage were measured.

Results

Infarct size was significantly smaller in diabetes. Moderate HG increased neuronal damage in diabetic but not in control rats. HT frequency and hemoglobin was significantly higher in diabetes. HG augmented vascular damage in control rats but had no additional effect on bleeding in diabetes. There was significant neurological deficit in diabetes which was worsened by HG.

Conclusions

Even moderate hyperglycemia worsens outcome from ischemic stroke. While moderate HG does not increase infarct size in control rats, it is sufficient to induce HT. A further elevation in blood glucose in diabetic animals does not worsen HT suggesting that diabetic animals already suffer from increased vascular damage.
Effect of skeletal muscle ischemia‐reperfusion (I/R) postconditioning (Post C) on antioxidant status in rats
Authors: Dubick Michael A , Mase Vincent J , Barr Johnny L , Grubbs Dana L , Roe Janet L , Walters Thomas J
Published: June 2021

Although tourniquets (TK) have reduced battlefield mortality, TK‐induced I/R has contributed to muscle loss. Methods to reduce associated muscle damage would be of great benefit. The present study investigated antioxidant status in skeletal muscle from rats after Post C in an established rat hindlimb TK model. Rats (n=10/gp) were randomly assigned to sham controls or 2 Post C conditions (either 4 or 8 fifteen sec cycles of reocclusion/reperfusion) immediately after 3h TK application. Rats were euthanized 24 hr later and TK‐muscle and the contralateral control muscle (C) were collected and frozen at −80o C until assayed. Tissues were analyzed for antioxidant potential, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), xanthine oxidase (XO), nitric oxide, myeloperoxidase (MPO) activity and antioxidant enzyme activities (GSH‐reductase, GSH‐peroxidase, superoxide dismutase and catalase). XO activity and total nitric oxide levels were about 30% and 25% higher, respectively, in all TK‐muscle compared to C muscle in all groups. MPO was also higher in TK than C muscle. Total antioxidant potential, TBARS, GSH and antioxidant enzyme activities were not different among groups in TK and C muscle. Taken together these data do not suggest that Post C improves antioxidant status in muscle injured by 3 hr TK application.
Concentration‐dependent Effects of Testosterone on Glial Cells Undergoing Oxygen/Glucose Deprivation/Reperfusion
Authors: ZHOU Jingyi , CHEN Najiao , SHEN Jia , JIN Weiwei , BRUCE Iain C. , XIA Qiang
Published: June 2021

To determine the effects of testosterone (T) on glial cells suffering from oxygen/glucose deprivation/reperfusion (OGD/RP) and the possible mechanisms, we randomly divided the cultured glial cells into sham group, control group and T groups which were pretreated for 4 h with T of (in nmol/L) 0.01, 0.1, 0.5, 1, 2, 5, 10, 100, 103, 104, 2×104, 5×104, 105 and 106 separately. 8 h OGD/24 h RP was performed. Trypan blue, xanthine oxidase, Fluo‐3/AM and BCECF/AM were used to evaluate survival rates, activities of superoxide dismutase (SOD), intracellular calcium ([Ca2+]i) and pH (pHi). The survival rates showed two peak values at 5nmol/L and 2×104 nmol/L (79.1%±1.09%, 57.3%±1.21%, mean±SD, P<0.05 vs. control) while three valley values at 0.1 nmol/L, 103 nmol/L and 105 nmol/L (24.4%±1.19%, 26.5%±2.32%, 16.7%±5.30%, mean±SD, P<0.05 vs. sham). In 5 nmol/L and 2×104 nmol/L groups, lower [Ca2+]i, higher pHi and SOD activities were found (P<0.05 vs. control). In conclusion, the concentration‐dependent anti‐ischemic effect of T is multi‐mechanism mediated and the further study is needed to investigate the molecular target of such protection.
Glycogen Synthase Kinase 3β Inhibitor Protects Against Vascular Hyperpermeability Following Hemorrhagic Shock
Authors: Childs Ed W , Hunter Felicia A , Tharakan Binu , Sawant Devendra A , Smythe W. Roy
Published: June 2021

Hemorrhagic shock (HS) disrupts the endothelial cell adherens junctions leading to vascular hyperpermeability. β‐Catenin is an integral component of the adherens junction complex which helps to maintain barrier integrity by binding to adjacent cells via VE cadherin and intracellularly to actin cytoskeleton. We hypothesized that inhibition of GSK‐3β that phosphorylates β‐catenin would attenuate vascular hyperpermeability. Our objective was to test GSK‐3β specific inhibitor SB216763 against hyperpermeability following HS. In rats HS was induced by withdrawing blood to reduce the MAP to 40 mmHg for 60 minutes followed by resuscitation. Rats were given SB216763 ten minutes prior to shock. To study vascular permeability, the rats were injected intravenously, with FITC‐albumin and its flux across the mesenteric post‐capillary venules were determined using intravital microscopy. Sham group showed minimal extravsation of FITC‐albumin in the extravascular space. HS group showed significant and time dependent increase in FITC‐albumin extravasation (p<0.05). SB216763 (600μg/kg) significantly decreased HS‐induced FITC‐albumin extravasation (p<0.05). β‐Catenin phosphorylation by GSK‐3β is an important mechanism that regulates vascular hyperpermeability following HS. GSK‐3β inhibitors may have therapeutic potential against vascular hyperpermeability following hemorrhagic shock.
The Purinergic Agonists ATP and UTP Act Predominantly via P2Y Receptors in Human Vascular Endothelial Cells
Authors: Braun Andrew P , Raqeeb Abdul , Sheng Jianzhong
Published: June 2021

Purinergic agonists, such as ATP, can produce robust vasodilation, yet the specific receptor(s) (i.e. P2X or P2Y) responsible for these effects remain poorly characterized. In this study, we have examined the purinergic receptors mediating acute Ca2+ mobilization, membrane hyperpolarization and nitric oxide (NO) synthesis in cultured HUVECs (EA.hy926 cell line). Brief application of either ATP or UTP (2 μM) to single HUVECs produced comparable elevations in cytosolic Ca2+, membrane hyperpolarizations and acute NO synthesis. These responses were largely unaffected in the presence of the non‐selective P2X receptor antagonists PPADS (10 μM) and TNP‐ATP (1 μM). In contrast to ATP and UTP, the selective P2X receptor agonist α,β‐methyl ATP (2 μM) had little effect on cytosolic Ca2+, membrane potential or NO production. Collectively, these data suggest that the observed stimulatory actions of ATP and UTP in this cell system occur primarily via P2Y, and not P2X, receptors. A plot of the concentration‐dependent rise in cytosolic Ca2+ evoked by ATP and UTP revealed similar EC50 values for the two agonists (1–3 μM); this equipotent action of ATP and UTP further suggests that P2Y2 receptors are the predominant purinergic sub‐type. Finally, RT‐PCR analysis confirmed the presence of P2Y2 receptor mRNA in this human vascular endothelial cell model.

Research support to AP Braun was provided by the CIHR and HSF Alberta
Elderly women demonstrate an attenuated vasoconstrictive response during a cold pressor stimulus
Authors: Jarvis Sara , VanGundy Tiffany , Galbreath Melyn , Shibata Shigeki , Meier Rhonda , Vongpatanasin Wanpen , Levine Benjamin , Fu Qi
Published: June 2021

Animal studies have shown that adrenergic vasoconstriction is reduced in older females but not males. The purpose of this study was to compare sympathetic and vasoconstrictor responses to a cold pressor stimulus between elderly women and men to determine whether sex differences in adrenergic vasoconstriction exist in aged humans. Heart rate (HR), arm blood pressure (systolic, SBP; diastolic, DBP) and muscle sympathetic nerve activity (MSNA) were measured in 9 women and 10 men [age: 69±7 (SD) vs. 69±4 yrs, respectively] during baseline (BL), a 2‐min cold pressor test (CPT), and 3‐min of recovery. HR during CPT (71±8 vs. 68±11 beats/min, P=NS) and changes in HR from BL (ΔHR: 4±5 vs. 4±6 beats/min, P=NS) were the same between the sexes. MSNA responses to CPT (BL: 543±289 vs. 491±145 a.u./min; CPT: 1039±567 vs. 965±161 a.u./min, both P=NS) were similar between elderly women and men; however, women did not demonstrate as large of an increase in DBP compared to men (CPT: 84±25 vs. 98±14 mmHg, P=0.02; ΔDBP: 8±10 vs. 22±12 mmHg, P=0.04). SBP was similar between the groups (BL: 129±42 vs. 129±11 mmHg; CPT: 150±46 vs. 156±19 mmHg; both P=NS). These findings suggest that elderly women demonstrate an attenuated vasoconstrictive response to a CPT when compared to elderly men. It is likely that the attenuated vasoconstrictive response in women is due to a decrease in vascular smooth muscle sensitivity to α1‐adrenergic stimulation.
Vitamin C reduces AT1R and AT2R activation in the rostral ventrolateral medulla of hypertensive sucrose‐fed rats
Authors: Oliveira Thaís Galvão Cesar , Oliveira‐Sales Elizabeth Barbosa , Casarini Dulce Elena , Bergamaschi Cássia Toledo , Heimann Joel C , Campos Ruy Ribeiro , Dolnikoff Miriam Sterman
Published: June 2021

The aim of this study was to evaluate blood pressure (BP), renal sympathetic nerve activity (rSNA), protein expression and activation of AT1R and AT2R in the RVLM of control (C, n=22), sucrose‐fed (20% sucrose solution, 30 days; SC, n=24) and sucrose‐fed rats treated with vitamin C (150 mg/Kg/day, last 7 days; SCvC, n=22). Thiobarbituric acid reactive substance (TBARS) was measured in serum. Insulin, Ang II and glucose were measured in plasma. Protein expression was evaluated by western blot and the receptors’ activation was evaluated by ELISA in tissue slices. BP, rSNA, insulin, Ang II and TBARS levels increased (P<0.05) in SC compared to C. In SCvC, Ang II and insulin levels were similar to SC although, BP, rSNA and TBARS were diminished. AT1R protein expression in the RVLM was similar in all groups while there was a 74% increase (P<0.05) in AT2R protein expression in SC compared to C. In relation to C, an intense (P<0.05) AT1R (116%) and AT2R activation (417%) were found in SC. In SCvC, AT2R protein expression levels were 43% lower than that of SC and were similar to those of C. AT1R and AT2R activation were also 39% and 29% lower in SCvC compared to SC. These results suggest that AT1R and AT2R activation in RVLM is involved in rSNA and hypertension in sucrose‐fed rats. Supported by FAPESP.
Apocynin inhibits and its removal augments L‐type Ca2+ currents in coronary artery smooth muscle cells and ventricular myocytes
Authors: Ochi Rikuo , Gupte Rakhee S , Murayama Takashi , Kurebayashi Nagomi , Gupte Sachin A
Published: June 2021

Exogenously applied H2O2 augments L‐type Ca2+ current (ICa,L). NADPH oxidase (NOX) initiates sequential reactions to generate reactive oxygen species (ROS) including H2O2. We studied the regulatory role of NOX‐generated ROS on ICa,L by applying apocynin (APO), a NOX inhibitor, and diphenyleneiodonium (DPI), a flavoprotein inhibitor. Whole cell ICa,L was recorded from bovine coronary arterial smooth muscle (BCASMC), rat ventricular myocytes (RVMC) and HEK 293 cells transiently expressing human cardiac CaV1.2. APO (0.1–10 mM) rapidly and dose‐dependently inhibited ICa,L: 10‐mM APO decreased ICa,L to 27, 34 and 16% in BCASMC, RVMC and 293 cells, respectively, while DPI (10 μM) inhibited more slowly and less intensively (to 64% in RVMC). The inhibitory effects of both inhibitors were mainly additive. Surprisingly, washout of high concentrations of APO rapidly recovered ICa,L to slightly above control in BCASMC and increased to 150% of control with prolonged current decay in RVMC. ROS levels measured simultaneously with ICa,L by increase rate of CM‐H2DCF fluorescence were decreased by APO and markedly increased by its removal in BCAVSM and RVMC. Possible accumulation of NADPH during NOX inhibition by APO explains the increased generation of ROS. We conclude that decrease of ROS inhibits and its increase augments cardiovascular ICa,L and NOX is involved in the regulation of ICa,L. This study is supported by NIH grant #R01HL85352
The Effects of Pressure Overload on Cardiac Geometry and Function are Aggravated by Aging
Authors: de Boer Martine , van Deel Elza D. , van der Horst Gijsbertus T.J. , Hoeijmakers Jan H.J. , Duncker Dirk J.
Published: June 2021

The aging heart undergoes structural and functional alterations, which are reminiscent of changes observed in the failing heart, and may thus increase the vulnerability of the aging heart to develop heart failure in response to mechanical overload. To investigate the effect of aging on left ventricular (LV) remodeling and dysfunction we subjected mice to pressure overload by transverse aortic constriction (TAC) at 3 and 24 months of age. Eight weeks after TAC, LV geometry, LV function and heart as well as lung weights were measured. Aging minimally affected cardiac geometry and function in sham operated animals. TAC not only induced LV hypertrophy, but also reduced fractional shortening in both age groups. However, only in 24 months old mice, TAC induced LV dilation from 3.5±0.2 mm in Sham mice to 4.4±0.2 mm in TAC mice and systolic dysfunction, demonstrated by a decrease in the rate of rise of LV pressure at 40 mmHg (dP/dtP40; Sham 7910±460 vs. TAC 4940±360 mmHg/s) as well as diastolic dysfunction evidenced by a decrease in dP/dtmin (Sham −6810±430 vs. TAC −5025±462 mmHg/s) and an increase in LV end diastolic pressure from 5±1 mmHg in Sham mice to 12±3 mmHg in TAC mice (all p<0.05). In conclusion, the effects of pressure overload on cardiac geometry and function are aggravated by aging.
Cardiac‐specific Cre‐recombinase causes a dose‐dependent transient reduction in systolic function
Authors: Smith Grant H , Hall Michael E , Stec David E , Hall John E
Published: June 2021

The Cre‐loxP system is a useful tool to study the physiological effects of gene knockout. We have employed a widely used inducible cardiac‐specific model, α‐Myosin Heavy Chain Mer‐Cre‐Mer, using tamoxifen (TAM) to activate Cre‐recombinase (Cre). Cre positive C57B/J6 mice were exposed to one (n=4), three (n=3), or five (n=7) daily doses of a 1 mg TAM injection. Echocardiography demonstrated no statistically significant differences in systolic function (SF) at baseline as assessed by ejection fraction (EF), fractional shortening (FS), stroke volume, or cardiac output. In mice exposed to five injections, a fall in all determinants of SF was observed 6 days after initiating TAM, but only EF and FS were decreased significantly. SF returned to near baseline measures 10 days after TAM initiation. Heart weight to body weight ratios were 4.45±0.34, 5.05±0.05, and 6.33±0.61 for one, three, and five TAM injections respectively. TAM had no effect on cardiac function in Cre‐negative mice. Thus, an inducible cardiac specific Cre caused a transient decline in SF that was dependent on the number of TAM doses.
Histone deacetylase inhibition (HDACi) attenuates catecholamine‐induced impairments in contractile reserve
Authors: Liles John Thomas , Joly Kristin M. , Ida Kevin K , Chapo Joseph , Plato Craig F.
Published: June 2021

Chronic catecholamine excess is a well‐characterized model of cardiac hypertrophy and dysfunction. We recently demonstrated adult, non‐senescent rats have impaired contractile reserve, and increased hypertrophic gene expression and caspase activity following chronic isoproterenol (ISO) administration. HDACi is reported to decrease stress‐induced cardiac hypertrophy, hypertrophic gene expression, and cell apoptosis. We evaluated the efficacy of Scriptaid, a hydroxamate pan‐HDACi, to inhibit impairments in contractile reserve following chronic ISO. Rats were administered Scriptaid (10 mg/kg, i.p.) or vehicle (100% DMSO) daily, followed 1 hr later by ISO (0.1 mg/kg, s.c.) or its vehicle for 3 wks. Chronic ISO impaired contractile reserve (Δ+dP/dtmax) to milrinone (20 – 200 μl/kg/min) by >50%, increased left ventricular (LV) collagen1a (13 fold) and ANF (5 fold) mRNA expression and caspase‐3 activity (24%), while inducing LV hypertrophy (33±10%). Scriptaid completely prevented decreases in contractile reserve and attenuated ISO‐induced LV hypertrophy by 37%. Scriptaid treatment reduced ISO‐induced increases in collagen1a and ANF mRNA expression (−53% and −33%, respectively) while normalizing caspase‐3 activity. These results suggest HDACi modulates epigenetic and apoptotic pathways thus improving cardiac performance in a model of catecholamine‐induced cardiac contractile dysfunction.
Cardiovascular Effects of a Nebulized B‐agonist Compared to Saline in Healthy Humans
Authors: Wong Eric C. , Wheatley Courtney M. , Cassuto Nicholas A. , Foxx‐Lupo William T. , Patanwala Asad E. , Snyder Eric M.
Published: June 2021

The β2‐adrenergic receptors (ADRB2s) are located in the heart and blood vessels. We sought to determine the influence of an inhaled β‐agonist on cardiac output (Q), heart rate (HR), stroke volume (SV), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and systemic vascular resistance (SVR) in healthy subjects (n=17, age=27±9years, ht=170±8cm, wt=68±12kg, Vo2Peak=98±27% predicted, mean±SD). To determine this, we assessed cardiovascular function before and 30, 60, and 90 minutes following the administration of a β‐agonist (2.5 mg albuterol in 3ml saline) or placebo (saline). Inhalation of saline resulted in no change in any of the measured or calculated cardiovascular parameters. Albuterol resulted in an increase in Q and SV, no change in HR or BP, and a drop in SVR (Q=3.6±1.0, 4.2±1.1, 4.4±1.3, and 4.3±1.1 liters/min.; HR=77±9, 80±10, 78±9, and 78±11 beats/min.; SV=46±12, 51±15, 56±14, 56±13ml/beat; SBP=106±12, 105±13, 107±10, and 108±10mmHg; DBP=69±8, 66±8, 69±8, and 68±9mmHg; MAP=80±11, 79±8, 81±7, and 81±9mmHg; SVR=1661±453, 1401±432, 1393±424, and 1384±391 dynes*sec/cm5, mean±SD, for baseline, 30, 60, and 90min post β‐agonist, p‐ANOVA<0.05 for Q, SV and SVR). These results demonstrate that the administration of a nebulized β‐agonist results in enhanced ventricular function and a drop in systemic vascular resistance, suggesting peripheral vasodilation.
Ob/Ob Mice But Not High Fat Fed Mice Exhibit Divergent Changes in Atrogin‐1 and Bnip3 Expression in Skeletal Muscle
Authors: Gaugler Megan , Reynolds Thomas H.
Published: June 2021

The purpose of this study was to determine whether diet‐induced insulin resistance and type 2 diabetes are associated with an increase in the expression of the muscle atrophy related genes atrogin‐1 and Bnip3. Mice were fed a high fat diet (HFD) or a normal chow (CON) diet for 8 months. Following the dietary intervention muscles were removed and processed for immuno‐blotting or quantitative polymerase chain reaction (qPCR) experiments to assess atrogin‐1 and Bnip3 protein and mRNA levels, respectively. We observed no effect of the high fat diet on atrogin‐1 and Bnip3 protein levels or mRNA levels. These results indicate that long‐term high fat diet interventions do not activate ubiquitin‐dependent protein degradation and/or autophagy. Identical experiments were conducted with muscles from genetically obese (Ob/Ob) mice. There was a significant decline in atrogin‐1 protein levels in muscles from Ob/Ob mice compared to CON mice. Furthermore, a trend existed for higher levels of Bnip3 expression in muscles from Ob/Ob mice (P = 0.078). These results suggest that the loss of muscle mass with severe type 2 diabetes is due, in part, to increased autophagy rather than ubiquitin mediated proteolysis. Alternative, a decline in atrogin‐1 levels in muscles from type 2 diabetics may lead to an accumulation of damage proteins resulting in skeletal muscle contractile dysfunction.
NADPH Oxidase Inhibition Alleviates Diabetes‐Induced Myocardial Contractile Dysfunction
Authors: Roe Nathan , Thomas Paul , Ren Jun
Published: June 2021

Reactive oxygen species are implicated in cardiac dysfunction in a variety of diseases including diabetes. Upregulation of the pro‐ROS enzyme NADPH oxidase (NOX) has been shown in diabetes and contributes to diabetic cardiac dysfunction. This study was designed to evaluate the effect of NADPH oxidase inhibitor apocynin on STZ–induced diabetic cardiac dysfunction. Experimental diabetes was induced in male C57 mice using STZ (150mg/kg) injection prior to administration of apocynin (4mg/kg/d for 2 wks in drinking water). Myocardial function was evaluated by echocardiography and edge‐detection. STZ treatment reduced fractional shortening, peak shortening, shortening/relengthening velocities, and systolic duration, all of which were attenuated or ablated by apocynin. There was no significant difference in systolic, diastolic or mean blood pressure among all groups tested. Apocynin treatment did not reverse the STZ‐induced loss in body weight or hyperglycemia. Western blot analysis revealed that apocynin failed to affect phosphorylation of the downstream insulin effector Akt, and dephosphorylation of AMPK. Apocynin did not affect STZ‐induced decrease in posterior wall thickness, suggesting a beneficial effect independent of cardiac remodeling. Taken together, our data indicated that inhibition of NADPH oxidase rescues diabetes‐induced myocardial dysfunction.
The Regulation of Cytokine Gene Expression by Sphingosine 1‐Phosphate in Endothelial Cells
Authors: Rossi Laura , Kovala Tom
Published: June 2021

Angiogenesis is the formation of new blood vessels from pre‐existing vasculature. Signaling mechanisms initiated by vascular endothelial growth factor (VEGF) and the lipid bioactivator of angiogenesis, sphingosine‐1‐phosphate (S1P) have been linked in recent studies. In human umbilical vein endothelial cells (HUVECs) stimulated with either VEGF or S1P, Janus kinases (JAKs) become activated and stimulate downstream signal transducers and activators of transcription (STAT) proteins that affect gene expression. The S1P signaling to JAKs is mediated by the transactivation of VEGF receptors. Given that VEGF and S1P are strong inducers of angiogenesis and that the JAK/STAT pathway mediates gene expression, an initial expression profile of 27 soluble growth factors and cytokines in S1P‐ and VEGF‐treated HUVECs was elucidated using Bio‐Plex technology. Treatment with either VEGF or S1P induced increased expression of the proangiogenic and proinflammatory factors IL‐6, IL‐8 and MCP‐1. Expression of an additional subset of 13 factors was increased with VEGF alone while S1P did not induce expression of any unique genes. This upregulation is dependent on treatment dose. While the extracellular matrix (ECM) that cells were grown on did not induce expression, specific ECM proteins did affect expression levels in treated cells. These findings reveal a potential role for JAK/STAT signaling in the angiogenic response.
TRPC6 and TRPC1 functionally interact to mediate Ca2+ entry in endothelial cells to induce lung vascular permeability
Authors: Tauseef Mohammad , Knezevic Nebojsa , Kini Vidisha , Vogel Stephen , Birnbaumer Lutz , Malik Asrar , Mehta Dolly
Published: June 2021

Transient receptor potential channels (TRPC6) is shown to mediate Ca2+ entry independent of ER store depletion by DAG, and is therefore called receptor operated channels (ROC). TRPC1 mediates Ca2+ entry in response to endoplasmic reticulum (ER) store depletion by IP3, and represents the store operated calcium channels (SOC). The prevailing view is that TRPC6 and TRPC1 function in an independent manner. We tested the hypothesis that TRPC6 and TRPC1 interact to regulate the rise in intracellular Ca2+ and thereby increase pulmonary microvessel permeability. Pulmonary endothelial cells isolated from TRPC6 or TRPC1 null mice failed to increase cytosolic Ca2+ in response to thrombin. We show that both TRPC6 and TRPC1 null mice do not develop pulmonary edema in response to thrombin which activates both ROC and SOC. Next, we used thapsigargin or DAG analog OAG which directly activates SOC or ROC to delineate the intricacies between TRPC6 and TRPC1 regulation of Ca2+ entry. We show that thapsigargin attenuated Ca2+ entry that rapidly decayed in TRPC1 null cells, whereas it did not significantly increase Ca2+ entry in TRPC6 lacking cells. Consistently we show that thapsigargin failed to induce detectable increase in microvessel permeability in TRPC1 or TRPC6 null mice lungs. Loss of TRPC6 prevented OAG induced Ca2+ entry and lung microvessel permeability. Deletion of TRPC1 had modest effect on OAG induced Ca2+ entry as well as pulmonary microvessel permeability. Our data indicate that TRPC6 and TRPC1 co‐ordinate to mediate SOC dependent Ca2+ entry in endothelial cells to signal increased lung microvessel permeability.
Nerve‐induced smooth muscle to endothelium signaling in small resistance arteries
Authors: Nausch Lydia Waltraud Maria , Bonev Adrian D , Tallini Yvonne , Kotlikoff Michael I , Nelson Mark T
Published: June 2021

Communication from endothelial cell (EC) to vascular smooth muscle cell (VSMC) has been shown to play a pivotal role in the maintenance of vascular tone. Despite emerging evidence that the converse pathway ‐ signaling from VSMCs to ECs ‐ may be important for feedback regulation of arterial diameter, the effects of nerve stimulation on EC calcium (Ca2+) dynamics are not known. Utilizing a transgenic mouse model in which the Ca2+ biosensor, GCaMP2, is expressed specifically in vascular ECs, we investigated the novel concept of nerve/VSMC to EC signaling. Here, we present the first evidence that electrical field stimulation (EFS) of sympathetic nerves causes a dramatic elevation of local EC Ca2+ signals (“pulsars”) in endothelial projections to SM as well as in Ca2+ waves in small resistance mesenteric arteries. Blocking VSMC purinergic and adrenergic receptors differentially affects the time course of the EFS‐induced elevation in EC Ca2+, suggesting that the early response to EFS requires activation of SM P2X1Rs and the delayed response requires SM alpha adrenergic receptors. Inhibition of EC intermediate conductance, calcium‐sensitive K+ (IK) channels enhances nerve‐evoked vasoconstriction. These data are consistent with a novel negative feedback mechanism whereby sympathetic nerve stimulation increases EC Ca2+ and IK channel activity to oppose SM activation. (Supported by the NIH).
p38 MAPK‐dependent MMP regulation during coronary collateral growth
Authors: Dodd Tracy y , Jadhav Rashmi , Smith Erika , Rocic Petra
Published: June 2021

We have previously shown that repetitive ischemia (RI) stimulated coronary collateral growth (CCG) in healthy (SD) but not metabolic syndrome (JCR) rats, and required p38 MAPK (p38) activation. Extracellular matrix (ECM) remodeling is associated with CCG, and regulated, in part, by matrix metalloproteinases (MMPs). Thus, we investigated the role of p38 in the regulation of MMP expression and activation in SD vs. JCR rats. An occluder was implanted over the LAD, and the rats underwent the RI protocol (8 40 sec occlusions; every 20 min, every 8 hrs for 0, 3, 6, or 9 days). CCG was measured in the ischemic, collateral‐dependent (CZ) and the normal (NZ) zones using microspheres, p38 activation and MMP expression by Western blot, MMP activity by zymography, and ECM remodeling by immunohistochemistry. SB203580 was given IV to specifically block p38 activation. Expression and activation of MMP1, 2 and 9 were increased on days 3 and 6 of RI in the CZ of SD rats. p38 inhibition decreased MMP 2 and 9 activation. In contrast, RI did not alter expression or activation of MMP1, 2 or 9 in JCR rats. MMP activation correlated with reduced elastin, laminin and type III collagen content. Thus, CCG is dependent on p38‐dependent regulation of MMP expression and activation. Furthermore, compromised CCG in the metabolic syndrome may be partially due to the lack of p38‐dependent MMP activation, and resultant decreased ECM remodeling. NIH RO1 HL093052.
Essential barrier functions of scar‐forming reactive astrocytes
Authors: Sofroniew Michael V
Published: June 2021

Astrocytes exert many functions in the healthy central nervous system (CNS) and respond to CNS insults with a process called reactive astrogliosis, which in its extreme form leads to scar formation. The reactive astrocyte scar has long been regarded as uniformly detrimental to clinical outcome after CNS injury because of its inhibition of axon regeneration. Over the past twelve years, transgenic mouse models have enabled investigation of the mechanisms and functions of reactive astrocyte scar formation in vivo. Surprisingly and unexpectedly, these studies show that experimental disruption of reactive astrocyte scar formation leads to a pronounced increase in the spread of inflammatory cells, greater neurodegeneration and demyelination, and greater loss of function after a variety of CNS insults. These findings indicate that reactive astrocyte scar formation provides essential neuroprotective barrier functions that restrict the migration of inflammatory cells after CNS trauma, infection and autoimmune disease. The blockade of axon regeneration by reactive astrocyte scars may be a secondary byproduct resulting from the redundancy across different cell types of molecular cues that inhibit cell migration. Therapeutic strategies to promote axon regeneration by blocking such inhibitory cues will require evaluation for potential effects on inflammatory and other cell types.
Adenosine A3 Receptor Agonists (A3RA) Induce Favorable Alterations in the MMP/TIMP Response in Skeletal Muscle Following Traumatic Injury
Authors: Urso Maria Laina , Wang Ruibo , Zambraski Edward J , Liang Bruce T
Published: June 2021

Our prior work identified robust changes in the expression of the Matrix Metalloproteases (MMPs) & their inhibitors (TIMPs) 3–72 h post‐injury in skeletal muscle. Dramatic upregulation of the MMPs is hypothesized to interfere with proper skeletal muscle regeneration. Because adenosine can provide protection in cardiac muscle, we evaluated the effects of an A3RA (CI‐IB‐MECA), on MMP & TIMP expression in skeletal muscle. Sixty mice were injected with Evans blue dye (EBD) & the A3RA, or a placebo (P) 2 h pre‐injury. Injury was induced by applying a cold (−20C) steel probe to the tibialis anterior (TA). TA samples were collected from injured & contralateral legs 3, 10, 24, 48, & 72 h post‐injury. qRT‐PCR was used to quantify mRNA. EBD analysis 24 h post‐injury indicated a trend (p=0.06) towards reduced percentage of injured cells in A3RA‐treated mice versus P‐treated (42% vs. 57%). A3RA‐treatment significantly (p<0.05) modulated the response of MMP‐2 (A3RA: no change (NC) vs. P: 9‐fold decrease); MMP‐3 (A3RA: 13‐fold vs. P: 17‐fold increase); MMP‐9 (A3RA: 2‐fold vs. P: 23‐fold increase); MT1‐MMP (A3RA: NC vs. P: 25‐fold decrease); TIMP‐1 (A3RA: 4‐fold vs. P: 27‐fold increase); & TIMP‐2 (A3RA: 2‐fold increase vs. P: 6‐fold decrease). These data suggest that the A3R is a novel target for ameliorating skeletal muscle injury through favorable alterations in the MMP/TIMP response.
Rapamycin (Sirolimus)–induced protection against ischemia‐reperfusion injury is mediated through AMPK, Akt and JAK/STAT pathways in mouse heart
Authors: Das Anindita , Salloum Fadi N , Xi Lei , Mayton Eric , Desai Shivani K , Ockaili Ramzi A , Kukreja Rakesh C
Published: June 2021

Rapamycin (RAPA) is an approved drug to prevent rejection of transplanted organs and coronary restenosis. We reported that RAPA induces protection against ischemia‐reperfusion (I‐R) injury in the heart and cardiomyocytes. For determining a causative role of survival kinases (AMPK, Akt and JAK/STAT) in RAPA‐induced cardioprotection, adult male ICR mice were pretreated with RAPA (0.25 mg/kg, i.p.) or vehicle (DMSO). Hearts were isolated after 30 min and subjected to 20 min global I and 30 min R in Langendorff mode. RAPA significantly reduced infarct size as compared to DMSO (from 33.5±1.7% to 9.4±1.2%, n=7/group; p<0.001). This protection was abolished by AMPK inhibitor, compound c (20 mg/kg, i.p., 39.4±2.2%), PI3K inhibitor, wortmannin (15 μg/kg, i.p., 29.3±4.8%), JAK2 inhibitor, AG‐490 (40 mg/kg, i.p., 37.6±5.1%) and STAT3 inhibitor, stattic (20 mg/kg, i.p., 33.3±1.2%). Western blots revealed that RAPA induced phosphorylation of AMPK, Akt, STAT3 and GSK3β and enhanced the Bcl‐2/Bax ratio in the heart as compared to DMSO. These studies provide new evidence that activation of AMPK, Akt, JAK/STAT, and inactivation of GSK3β play critical role in RAPA‐induced protection against I‐R injury in the heart. (Supported by AHA BGIA 0765273U to AD and NIH HL51045, HL79424, HL93685 to RCK)