bluuebunny/crossref_metadata_2025 huggingface dataset:
Ric‐8A binds to and regulates the activation state of the RGS14:Gai1‐GDP signaling complex
Authors: Vellano Christopher P. , Shu Feng‐jue , Ramineni Suneela , Yates Cindee , Tall Gregory G. , Hepler John R.
Published: June 2021

We have shown RGS14 to be a scaffolding protein in brain that binds Gai, H‐Ras, and Raf kinases to regulate MAPkinase signaling. Like other RGS proteins, RGS14 acts as a GTPase activating protein (GAP) to terminate Gai/o signaling. Unlike other RGS proteins, RGS14 contains a GPR/GoLoco (GL) domain that binds inactive Gai1/3‐GDP in cells and in vitro. Here we provide evidence that RGS14 acts as a GL protein in unconventional G protein signaling, independent of G protein‐coupled receptors (GPCRs). Consistent with this idea, we find in cells that RGS14 binds Ric‐8A, a non‐GPCR guanine nucleotide exchange factor (GEF) that regulates other GL:Gai‐GDP complexes. When co‐expressed with Gai1‐GDP in cells, both RGS14 and Ric‐8A are recruited from the cytosol to the plasma membrane, suggesting formation of a protein complex. Ric‐8A induces dissociation of the RGS14:Gai1‐GDP complex in cells and in vitro. This dissociation allows Ric‐8A to act as a GEF towards Gai1; however, RGS14 inhibits the stimulatory effects of Ric‐8A on the GTP binding and steady‐state GTPase activity of Gai1. Native RGS14 and Ric‐8A co‐localize to the same hippocampal neurons, further supporting a functional link between these proteins. These findings indicate that Ric‐8A is a novel regulator of the RGS14:Gai1 signaling complex. Studies are underway to determine how MAPkinase signaling modulates Ric‐8A effects on RGS14/Gai1 signaling in brain.
Modulation of Response to Stress by Estradiol Benzoate and Selective Estrogen Receptor Agonists
Authors: Sabban Esther Louise , Harris Heather A , Serova Lidia I
Published: June 2021

Pretreatment with estradiol benzoate (EB) was found to modulate response of HPA axis and gene expression in several catecholaminergic neuronal locations in OVX female rats exposed to single immobilization (1xIMO) stress. Here we examined ER subtypes involved using selective agonists for ERα (PPT) or ERβ (WAY‐200070) on response to 1xIMO and repeated (6xIMO) stress. OVX female rats received 21 daily injections of either EB (25 μg/kg), PPT (10 μg/kg), WAY‐200070 (10 μg/kg) or vehicle. EB and PPT, but not WAY‐200070, reduced body weight by about 20% and uterine weight 8‐fold Both EB and PPT increased plasma corticosterone about 2–3 fold, but prevented any further rise with either 1xIMO or 6xIMO, indicating an ERα mediated mechanism. In the locus coeruleus (LC), the rise in DBH mRNA with both stress paradigms was abrogated with EB or PPT, while, WAY‐200070 blocked the response of DBH mRNA to 1xIMO but not 6xIMO. In the nucleus of solitary tract (NTS), the rise in TH and DBH mRNAs with 1xIMO or 6xIMO was absent, or greatly attenuated in EB or PPT treated animals. In most cases, WAY‐200070 inhibited the response to single but not repeated IMO. The results demonstrate that pretreatment with estradiol, or ER selective agonists, modulate the stress triggered induction of gene expression of NE biosynthetic enzymes in LC and NTS, with ER selectivity depending on the duration of the stress. (NIH grant NS 28869 by Wyeth Research)
ACETYLSALICYLIC ACID ENHANCES PURINERGIC RECEPTOR‐MEDIATED OUTWARD CURRENTS IN RAT MEGAKARYOCYTES
Authors: Young Jose Pompilio , Beckerman Jacob , Vicini Stefano , Myers Adam
Published: June 2021

Megakaryocytes (MKs) as platelet progenitor cells are a relevant model for platelet reactivity. Patch clamp techniques were used to analyze effects of acetylsalicylic acid (ASA), a COX‐1 inhibitor antithrombotic agent, on purinergic receptor‐mediated Ca++ activated K+ outward currents in rat MKs. Bone marrow MKs were incubated in 1mM ASA (ASA and sodium salicylate IC50 plasma value 2.5 mM; USP Convention. 2000). Using a K+ rich internal solution, currents were recorded in response to 10 μM ATP, 10 μM ADP or 5 μM 2MeSADP in the voltage clamp mode. Agonist‐induced currents decreased in amplitude over time, but this decline was attenuated by ASA in both continuous and repeated agonist challenge, indicating increased MK reactivity. In different cells, heterologous desensitization was observed when MKs were stimulated with ADP after exposure to a thromboxane receptor agonist (U46619), indicating cross‐talk between thromboxane and purinergic pathways. Different cells, treated with ASA or MRS2179 (P2Y1 receptor blocker), were stimulated with 2MeSADP. The dose response curve to 2MeSADP was shifted to the left by both drugs, suggesting increased MK reactivity. Current amplitude was increased by 30 μM IP3 added to the internal solution; this was also attenuated by ASA, suggesting that ASA might affect the P2Y‐IP3‐calcium signaling pathway. These findings may be relevant to the increasingly reported ASA resistance during clinical treatment.
Polymicrobial sepsis and endotoxemia promote microvascular thrombosis via distinct mechanisms
Authors: Patel Kavita , Soubra Said H , Lam Fong W , Rumbaut Rolando E
Published: June 2021

Objective

To determine whether the clinically relevant model of polymicrobial sepsis induced by cecal ligation/perforation (CLP) induces similar responses via the same mechanisms as endotoxemia.

Methods

We used a light/dye injury model in the cremaster microcirculation of C57BL/6 (wild type), and mice deficient in either toll‐like receptor‐4 (C57BL/10ScNJ) or vWF. Mice underwent CLP or sham surgery (six hours prior to experiments); in other groups, mice received an intraperitoneal injection of endotoxin (LPS) or saline (four hours prior to experiments).

Results

Both CLP and LPS enhanced thrombotic occlusion in wild type mice; however, in contrast to LPS, CLP also enhanced thrombosis in TLR4‐ and vWF‐deficient strains. LPS, but not CLP, increased plasma vWF antigen relative to controls. Septic mice, particularly those undergoing CLP, developed significant hemoconcentration. Intravenous fluid replacement with isotonic saline prevented the hemoconcentration and the prothrombotic responses to CLP, though fluids did not prevent the prothrombotic response to LPS.

Conclusions

Polymicrobial sepsis induced by CLP and endotoxemia promote microvascular thrombosis via distinct mechanisms; enhanced thrombosis induced by CLP is independent of TLR4 and vWF. The salutary effects of intravenous fluid replacement on microvascular thrombosis in polymicrobial sepsis remain to be characterized.
ICAM‐1 is Key Molecule in Carbamylated LDL‐induced Monocyte Adhesion
Authors: Apostolov Eugene , Ray Debarti , Savenka Alena , Shah Sudhir , Basnakian Alexei
Published: June 2021

Carbamylated LDL (cLDL) is a strong proatherogenic factor that is associated with chronic renal failure (CRF). It causes endothelial toxicity and exacerbates monocyte adhesion to endothelial cells. Monocyte adhesion is a key atherogenic process that leads to extensive housing of macrophages and formation of foam cells filled with lipids, the major marker and driving mechanism of atherosclerotic plaque formation. Recently, we showed that ICAM‐1 was the most employed molecule for cLDL‐mediated monocyte in vitro. Here we explored whether ICAM‐1 mediates the cLDL‐induced adhesion to vascular endothelium in in vivo model. For this we utilized the animal CRF model and urea‐consuming (UC) model in Apolipoprotein E knockout (KO) mice. Our results suggest that cLDL is elevated in sera and aortic walls of CRF and UC mice as compared to control groups. ICAM‐1 expression and number of housed macrophages in aortic wall and atherosclerotic plaques are significantly increased in experimental animals. Finally, monocyte cLDL‐induced adhesion was studied in ICAM‐1 KO mice. Our data suggest that cLDL induces significantly higher monocyte adhesion in wild type mice as compared to ICAM‐1 KO. Taken together, our data suggest that ICAM‐1 is a leading molecule that mediates mechanism of cLDL‐induced monocyte adhesion. The study was supported by AHA grant to E.A. and NIH/NHLBI R21 to A.B.
Antioxidant and anti‐inflammatory properties of apo A‐I milano
Authors: Purushothaman Meerarani , Puru Priya , Cimmino Giovanni , Badiom Juan J
Published: June 2021

The apolipoprotein A‐I Milano (ApoA‐I Milano), the mutant form of ApoA‐I was found to reduce plaque size and composition in an experimental model and enhanced reverse cholesterol transport evident primarily in HDL. We hypothesize that ApoA1 Milano may also possess antioxidant and anti‐inflammatory properties and prevent atherosclerotic disease process. The mouse macrophage cell line J774A‐1 was incubated with apoA‐I Milano or HDL and exposed to oxLDL or TNF‐alpha. The oxidative stress was quantified by the dichlorodihydrofluorescein method, the DNA binding activity of NF‐kB was measured by ELISA and the interleukin‐6 gene expression by PCR. The oxidative stress induced by oxLDL was markedly reduced by apoA‐I Milano or HDL incubation. Similarly the increase in NF‐kB activity induced by oxLDL or TNF‐alpha was reduced by apoA‐I Milano and HDL. However, the induction of inflammatory cytokine IL‐6 gene by TNF‐alpha was markedly downregulated by apoA‐I Milano (P<0.0001) than by HDL (P<0.003). These results indicate apoA1 Milano may possess antioxidant and anti‐inflammatory properties. Our data suggest that apoA‐I Milano downregulates the oxidative and inflammatory response associated with atherosclerotic disease process. Furthermore this study indicates that apoA‐I Milano exhibited enhanced anti‐inflammatory mechanism compared to ApoA1/HDL.
Cellular Mechanism to Infection ‐ Induced Thrombotic Risk
Authors: Jayachandran Muthuvel , Sticha Benjamin J. , Miller Virginia M.
Published: June 2021

Little is known about mechanisms by which bacterial infection contributes to thrombotic risk. Experiments were designed to identify the relationship between toll‐like receptor 4 (TLR4) activation by bacterial lipopolysaccharide (LPS) and circulating procoagulant (phosphatidylserine (PS) positive) microvesicles (MV). Blood from wild type (WT), and TLR4‐ gene deleted (dTLR4) female mice was collected prior to and following exposure to either saline or LPS 20ng/g body weight administered as a single intravenous injection (acute exposure) or after administration by subcutaneous osmotic pump (chronic exposure). MV were isolated from blood by differential centrifugation and identified by flow cytometry. MV thrombin generating capacity was measured by solid phase fluorescent assay. Procoagulant MV were significantly lower in dTLR4 compared to WT mice (P<0.007). Platelet derived MV were the most prominent MV in saline treated mice. Following LPS‐treatment, counts of procoagulant MV increased (P<0.006) significantly only in WT mice. The increase was greater after chronic compared to acute exposure. Thrombin generating capacity positively correlated (r2=0.9) with PS expression. These results suggest that increased procoagulant MV through TLR4 activation and thus explain in part increased thrombotic risk with bacterial infection. This work is supported by AHA‐SDG 30503Z and Mayo Foundation.
How blood brain barrier is breached in a mouse model of INCL
Authors: SAHA ARJUN , Munasinghe Jeeva , Zhang Zhongjian , Heffer Alison , Mukherjee Anil B
Published: June 2021

Infantile neuronal ceroid lipofuscinosis (INCL), a devastating neurodegenerative storage disorder of childhood, is caused by mutations in the palmitoyl‐protein thioesterase‐1 (Ppt1) gene. Using a mouse model of INCL, Ppt1‐knockout (Ppt1‐KO) mice, we recently reported elevated levels of pro‐inflammatory cytokines such as MCP1, IL‐1β, IL‐6 and TNFα. Since many of these parameters are associated with disruption of the blood brain barrier (BBB), we sought to determine: (a) whether BBB is disrupted in the Ppt1‐KO mice and if so, (b) what might be the molecular mechanism(s). Magnetic Resonance Imaging results showed that while in the 6‐month old Ppt1‐KO mice gadolinium (Gd, a contrast medium) readily infiltrated the brain parenchyma, such infiltration was not detectable in the WT mice indicating the BBB disruption in the Ppt1‐KO mice. We also found decreased levels of various tight‐junction proteins in the brain tissues of the Ppt1‐KO mice. A significantly higher level of IL‐17 as well as TH17 positive cells were found in the brain of the Ppt1‐KO mice. Moreover, the levels of activated matrix metalloproteinases (MMPs), which adversely affect the BBB, are significantly higher in the Ppt1‐KO mouse brain. Treatment of cultured glial cells with IL‐17 in vitro appears to stimulate MMPs expression. We propose that TH17‐mediated activation of matrix metalloproteinases disrupts the BBB contributing to INCL pathogenesis.
Response of the Murine Urinary Bladder Microcirculation to Lipopolysaccharide from Escherichia coli and Pseudomonas aeruginosa
Authors: Kowalewska Paulina , Burrows Lori , Fox‐Robichaud Alison E.
Published: June 2021

There is in vitro evidence demonstrating the inflammatory response to lipopolysaccharide (LPS) differs depending on the bacterial source. The objective of our study was to examine the in vivo inflammatory response to LPS from two common uropathogens, Escherichia coli (Ec) and Pseudomonas aeruginosa (Pa).

LPS or saline was injected transurethrally into bladders of female C57Bl/6 mice at varying doses. The bladder was exteriorized, and the bladder microcirculation examined by intravital microscopy. Leukocyte adhesion at 4 hrs post challenge with 5 mg/kg Pa LPS was 2.9±0.6 cells/field of view and at 7 mg/kg it was 3.9±0.8 cells/field of view, a significant increase from saline control. In contrast, leukocyte adhesion 4 hours post 5 mg/kg Ec LPS challenge was 17.4±3.9 and with 7 mg/kg 19.7±2.9 cells/field of view. However, at 4.5 hours adhesion was similar with either LPS (5 mg/kg) with 18.4±3.0 and 21.2±4.0 cells/field of view, respectively. The flux of rolling leukocytes was significantly increased above baseline levels only after Ec LPS challenge. There was also a significant increase in the number of neutrophils with Ec LPS stimulation.

In support of the in vitro observations, these in vivo results demonstrate that Ec LPS is more potent and induces a more rapid inflammatory response than Pa LPS.

Supported by a Collaborative Health Research Projects grant from CIHR and NSERC.
Monocyte adhesion and endothelial hyperpermeability are associated with failure of arteriovenous fistula (AVF) maturation for hemodialysis access
Authors: Lee Eugene S , Shen Qiang , Guo Mingzhang , Pitts Robert L , Wu Mack H , Yuan Sarah Y
Published: June 2021

Leukocyte‐endothelium interactions play an important role in vascular remodeling. The aim of this study was to test the hypothesis that monocyte adhesion‐induced endothelial hyperpermeability hampers vascular graft remodeling and maturation leading to AVF failure. Monocytes were isolated from 5 patients with successful AVF and 5 patients with failure of AVF maturation. The expression of adhesion molecules was measured using flow cytometry. In parallel, monocyte transendothelial migration and its associated endothelial permeability were assessed in a monocyte‐HUVEC co‐culture model. Compared to monocytes from the group with successful AVF, monocytes obtained from patients with AVF failure had significantly higher levels of CD11c expression and trans‐endothelial migration. Monocytes from patients with failed AVF induced a further decrease in transendothelial electric resistance. The data suggest that monocyte adhesion and endothelial hyperpermeability contribute to the failure of AVF maturation. This work was supported by R01 HL061507, HL084542, and a Peripheral Vascular Surgery Society Grant.
Tumor‐derived TIMP‐2 mediates endothelial barrier dysfunction and breast cancer cell transmigration by activating endothelial MMP‐2
Authors: Shen Qiang , Lee Eugene S , Pitts Robert L , Wu Mack H , Yuan Sarah Y
Published: June 2021

Accumulating evidence suggests a dual role of tissue inhibitor of metalloproteinase‐2 (TIMP‐2) in regulating the activity of matrix metalloproteinase‐2 (MMP‐2), which has long been associated with tumor angiogenesis and metastasis. By forming a complex with pro‐MMP‐2 and its cell surface activator MMP‐14, TIMP‐2 can either induce or restrain MMP‐2 activation. Our recent work has shown that breast cancer cell adhesion to vascular endothelial cells activates endothelial MMP‐2. The purpose of this study was to explore the underlying mechanism and its potential impact on endothelial barrier function and transendothelial migration. We found that MMP‐14 was expressed in both breast cancer cells (MDA‐231) and lung microvascular endothelial cells (HBMVEC‐L), whereas TIMP‐2 was exclusively expressed in cancer cells. Importantly, the tumor cell‐derived TIMP‐2 was identified as a major determinant of MMP‐2 activation during tumor cell transmigration in the presence of MMP‐14. This response was associated with endothelial barrier dysfunction, as evidenced by a significant decrease in transendothelial electric resistance and endothelial cell‐cell junction disruption. These findings suggest a cooperative mechanism between metastatic breast cancer cells and microvascular endothelial cells in promoting MMP‐2 activation and tumor cell transmigration. NIH grants HL61507, HL70753, HL73324, HL84852
Mechanisms of impaired acetylcholine‐mediated vasodilation in overweight rats
Authors: Lekic Mateja , Walker Benjimen R , Sweazea Karen L
Published: June 2021

The current study was designed to examine the early effects of increased adiposity in adult male Sprague‐Dawley rats fed either a high sucrose (HSD) or high fat diet (HFD) for 6 weeks. Initial studies examined pathways involved in acetylcholine (ACh)‐mediated vasodilation which is typically thought to act through increased production of the vasodilator nitric oxide (NO). Similar to obese animals, HSD and HFD‐fed rats developed impaired ACh‐mediated vasodilation in isolated mesenteric arteries compared to chow fed controls that was normalized by antioxidants. Unlike chow controls, inhibition of NO did not further impair vasodilation in the HSD or HFD groups suggesting that ACh may activate pathways distinct from NO in these settings. To examine smooth muscle sensitivity to NO, arteries were exposed to increasing concentrations of the NO donor sodium nitroprusside (10−11 to 10−3M). We found that HSD and HFD reduced smooth muscle sensitivity to NO. These data suggest that increased ROS scavenging of NO coupled with diminished VSM reactivity minimizes the role of NO in vasodilation following HSD or HFD which is partially compensated by activation of parallel vasodilatory pathways.
Retinal arteriolar endothelial dysfunction in early stage of diabetes in porcine model
Authors: Hein Travis W. , Potts Luke B. , Ren Yi , Kuo Lih
Published: June 2021

Hyperglycemia is implicated in retinal vascular dysfunction associated with the development of diabetic retinopathy. Although release of vasodilators from the endothelium of retinal arterioles is known to play an important role in the regulation of retinal blood flow, the impact of hyperglycemia on vasodilator reactivity of these microvessels remains unclear. Herein, using a streptozotocin‐induced type‐1 diabetes model in the pig, we examined nitric oxide (NO) synthase (NOS)‐ and cyclooxygenase (COX)‐mediated dilation of isolated retinal arterioles at 2 and 6 wk of hyperglycemia (485±64 mg/dL vs. 81±23 mg/dl in age‐matched control). In diabetic retinal arterioles, dilation to endothelial NOS‐mediated agent bradykinin but not to endothelium‐independent NO donor sodium nitroprusside or COX‐mediated agent forskolin was significantly reduced in a similar manner at 2‐ and 6‐wk hyperglycemia. Dihydroethidium staining showed markedly increased levels of superoxide in diabetic arterioles, while protein expression of NAD(P)H oxidase subunit p47phox was elevated. These findings show that acute hyperglycemia selectively impairs endothelial NOS‐mediated dilation of retinal arterioles in a diabetic pig model. Enhanced production of NAD(P)H oxidase‐derived superoxide likely contributes to the reduction of NO bioavailability. Support: EY018420 (TWH); Scott & White Research Foundation (LK).
Breast Cancer Microparticles Promote Formation of a Microenvironment Favorable for Lung Metastasis
Authors: Bauer Natalie Norwood , Rai Jyoti , Chen Hairu , Harris Lillianne , Shevde Lalita , King Judy
Published: June 2021

Microparticles (MPs) are 0.2–1 μm vesicles released from activated cells. Data suggest MPs from a human breast cancer cell line promote endothelial retraction necessary for metastasis. Recently it was reported that increased serum levels of activated leukocyte cell adhesion molecule (ALCAM) from breast cancer patients may serve as a biomarker for breast cancer. Our previous data showed that MPs derived from MDA‐MB‐231 breast cancer cells expressed ALCAM. We hypothesized that ALCAM‐positive MPs may help form a microenvironment for metastatic breast cancer cells. We “primed” the lung vasculature of rats with ALCAM‐positive MPs to determine if prior exposure to MPs could alter metastases. MPs were collected from MDA‐MB‐231 human breast cancer cells by differential centrifugation. Isolated rat lungs were perfused first with ALCAM‐positive MPs followed by perfusion with MDA‐MB‐231 cells for 90 min. Sections from the fixed lung tissue were examined. We found no change in the number of single breast cancer cells in the lung with MP priming. However, we observed a trend for increased tumor cells and cell clusters if ALCAM‐positive MPs are perfused prior to tumor cells. Cancers can release MPs prior to the release of intact tumor cells into the vasculature. This release of MPs may prime the vasculature in the lungs and promote metastases. AHA 065377B and P20 MD002314 (King); 0765413B and Parker B. Francis Fellowship (Bauer).
Intravenous Infusion of Keratose‐based Fluid Induces Arteriolar Vasodilation in Cremaster Muscle of Rats
Authors: Nunez Fiesky Alejandro , Trach Simon , Callahan Michael , Van Dyke Mark , Smith Thomas
Published: June 2021

INTRODUCTION

Synthetic plasma expanders have been investigated for treatment of Hypovolemic Shock. None have proven to be more beneficial than another or as effective as whole blood. Hyperosmotic hyperviscous Keratose is proposed as a possible plasma expander. This type of compound potentiates the release of strong vasodilators and induces net intravascular transport of water, increasing circulating volume. This results in peripheral vasodilation and increased cardiac contractility which translates into lower cardiac work and better tissue perfusion. The null hypothesis was tested that a Keratose resuscitation fluid (KRF) would not induce more arteriolar vasodilation than a current plasma expander, Hetastarch (HS).

METHODS

A topload of KRF, HS, or PBS was infused into euvolemic rats. Changes in arteriolar diameters were measured using intravital microscopy (Fig 1).

RESULTS

ANOVA showed significant differences (p <0.05) between the three treatments. Post‐hoc comparisons showed: KRF induced greater vasodilation than PBS or HS. HS vasodilatilatory effects were not significant compared to PBS (Fig 2).

DISCUSSION

KRF induced significant vasodilation in muscle microvasculature compared to HS or PBS. The use of KRF in a hemorrhage model could improve functional capillary density, a reliable index of tissue perfusion highly correlated with survival in severe hemorrhagic shock.
The aging rabbit heart as a model for cardiac aging
Authors: Cooper Leroy Leon , Odening Katja E. , Ziv Ohad , Chaves Lenny , Schofield Lorraine , Choi Bum‐Rak , Koren Gideon
Published: June 2021

Objective

To characterize of the effect of aging on His‐Purkinje and ventricular conduction.

Methods

Old (4 – 5.5 years) and young (5 – 8 months) female NZW rabbits were subjected to transvenous catheter‐based in vivo electrophysiological (EP) and hemodynamic studies. Mason Trichrome staining was used to evaluate differences in fibrosis.

Results

Among the old cohort we observed a cumulative incidence of sudden death (SD) of 5%. EP studies revealed significantly prolonged QRS complexes (old: 64.8ms ± 3.7 vs. young: 49.8ms ± 0.3, p=0.002) and prolonged His‐to‐Ventricle intervals in old rabbits (old: 31.9ms ± 1 vs. young: 23ms ± 1, p<0.0001), indicating a slowed conduction through the His‐ Purkinje. Also, older rabbit hearts were more easily VF‐inducible (7/8) than young hearts (3/12, p<0.05), indicating an underlying substrate for arrhythmias. Substantial slowing of conduction velocity (CV) was seen transverse to fiber orientation in old compared to young (0.2±0.03m/s vs 0.33± 0.07m/s; p<0.05). VF in old hearts demonstrated repetitive formation of lines of functional conduction block parallel to fiber orientation. Old hearts showed more pronounced fibrosis in the ventricle and the septum that appeared to infiltrate the myocardium from the epicardiaum towards the subepicardium.

Conclusion

Increase in anisotropic conduction due to fibrosis likely plays a key role in the pathogenesis of VF in old hearts.
Standing Test after the Blood Donation can Predict the Incidence of Late Phase Vasovagal Reaction
Authors: Yoshida Masayoshi , Dan Emi , Narita Sumito , Momii Hidetoshi , Kadokami Toshiaki , Andoh Shin‐ichi
Published: June 2021

Background

A major complication of donation is vasovagal reaction (VVR) with/without syncope. VVR occurs not only in the early phase but also in late phase after donation. Some risk factors have been reported but they have not satisfactorily predicted late VVR.

Methods and Results

After blood collection, 97 healthy donors were kept standing for 3minutes while blood pressure (BP) and heart rate (HR) were recorded. Questionnaires were returned from them to report late VVR during 24 hrs after donation. HR of donors with late VVR increased significantly compared with donors with no symptoms (Figure A). Area under the ROC curve about the HR after 2 minutes is 0.8 (Figure B).

Conclusion

HR response to standing after blood collection may satisfactorily detect the high‐risk donors prone to late VVR.
Increased capacity to oxidize fatty acids does not prevent development of hepatic steatosis in high‐fat diet fed rats
Authors: Ciapaite Jolita , van den Broek Nicole M.A. , Houten Sander M. , Wanders Ronald J.A. , Nicolay Klaas , Jeneson Jeroen A.L. , Prompers Jeanine J.
Published: June 2021

Objective

Assess adaptations of hepatic lipid metabolism in response to high‐fat diet (HFD).

Methods

Male Wistar rats (14 wk) were fed either normal chow (NC, 9 En% fat) or HFD (45.7 En% fat) ad libitum for 2.5 wk and 25 wk (n=10 per diet group). Intrahepatic lipids (IHL) were assessed using in vivo 1H magnetic resonance spectroscopy and Oil Red O staining. Fatty acids (FA) were quantified using gas chromatography. Mitochondrial function and DNA (mtDNA) copy number was assessed using high‐resolution respirometry and quantitative PCR, respectively.

Results

HFD feeding resulted in obese and glucose intolerant phenotype. Oil Red O staining showed that HFD feeding induced microvesicular steatosis. The content of IHL and FA increased with increasing duration of HFD feeding. The strongest effect was observed on the content of mono‐unsaturated FA (predominantly oleate), which may be due to increased activity of stearoyl‐CoA desaturase. HFD feeding stimulated mitochondrial biogenesis and oxidative FA metabolism as indicated by increase in mtDNA copy number and higher palmitoyl‐L‐carnitine‐driven oxygen consumption rate in state 3.

Conclusions

HFD feeding leads to accumulation of hepatic TAG despite of increased capacity to oxidize FA, indicating that the latter adaptations are insufficient to cope with oversupply of dietary lipids.

This work was financed by the Netherlands Consortium for Systems Biology.
Mechanisms of impaired insulin signaling in calcineruin A‐alpha knockout mice
Authors: Gooch Jennifer L , Roberts Tiffany K , Price Russ , Reddy Ramesh N
Published: June 2021

Post‐transplantation diabetes mellitus (PTDM) is a common adverse drug reaction resulting from long term use of calcineurin inhibitors (CIs). Development of calcineurin A subunit isoform specific knockout mice have played an important role in our understanding of the mechanisms of CI‐induced adverse effects including nephrotoxicity. However, the molecular mechanisms of CI‐induced PTDM are still unclear. Recently, our laboratory rescued the early lethality phenotype of calcineurin Aα−/− mice. Examination of blood glucose levels at weekly intervals until 8 weeks in these rescued mice revealed sustained hyperglycemia. This phenotype is unique to calcineurin Aα−/− mice and is not observed in calcineurin Aβ−/− mice. Insulin levels and insulin sensitivity tests indicated impaired insulin signaling as a cause for the pathogenesis of hyperglycemia. Membrane expression of the glucose transporter Glut4 was decreased in muscle tissue in calcineurin Aα−/− mice but there was no difference in total Glut4 levels. This suggests that calcineurin Aα may play a novel role in trafficking of Glut4. Next, primary myoblast cells isolated from wild‐type and calcineurin Aα−/− mice were treated with insulin to examine insulin signaling. Initial western blot analyses of cell lysates revealed significant decreases in the expression of both insulin receptor substrates IRS1 and IRS2 in calcineurin Aα−/− myoblasts. There was no difference in downstream phosphorylation of Akt in response to insulin in null cells. In contrast, insulin did not stimulate phosphorylation of ERK1 and 2 in calcineurin Aα−/− myoblasts. These results are particularly interesting in light of a recent report that CIs stimulate degradation of IRS proteins. In conclusion, the present study reveals a novel role for calcineurin Aα in insulin signaling and glucose homeostasis and may contribute to our understanding of clinical PTDM.
Central NPY deficiency does not enhance the chronic actions of melanocortin 3 and 4 receptors (MC3/4R) activation on glucose homeostasis, appetite and cardiovascular function in diabetic mice
Authors: Freeman John Nathan , do Carmo Jussara M. , Hall John E. , da Silva Alexandre A.
Published: June 2021

Acute studies suggest that NPY and MC3/4R exert opposite effects on appetite, cardiovascular and metabolic functions. This study tested if the responses to chronic MC3/4R activation on appetite, glucose levels (GLU), blood pressure (BP), and heart rate (HR) are altered in NPY deficient (NPY(−/−)) mice compared to wild‐type (WT) mice after induction of insulin‐deficient diabetes by streptozotocin (STZ) injection, an experimental model of diabetes associated with high central NPY levels. Despite lower baseline GLU compared to WT mice (139±6 vs 241±10 mg/dl), NPY(−/−) mice exhibited similar GLU 7 days after STZ injection (472±26 and 496±3 mg/dl). Food intake also increased similarly in both groups (3.7±0.2 to 7.0±1.0 and 3.6±0.3 to 6.0±1.4 g/day, respectively for NPY(−/−) and WT mice). No changes in HR or BP were observed 7 days after STZ injection (NPY(−/−) mice: 560±11 to 561±12 bpm and 113±2 to 116±4 mmHg; WT: 561±1 to 542±11 bpm and 103±3 to 108±2 mmHg). Infusion of the MC3/4R agonist MTII (120 μg/day, i.p. delivered by osmotic pumps) for 7 days did not significantly alter BP or HR in both groups, and did not attenuate the continuous rise in GLU (681±35 and 717±23 mg/dl) or food intake (9.8±1.4 and 9.2 0.3 g/day, respectively for NPY(−/−) and WT mice). Our results indicate that central deficiency of NPY does not exacerbate the chronic effects of MC3/4R activation to suppress appetite, to reduce GLU, or to increase BP and HR.
LUTEOLIN REDUCES CARDIAC DYSFUNCTIONS AND MITOCHONDRIAL OXIDATIVE STRESS IN STREPTOZOTOCIN‐INDUCED DIABETIC RATS
Authors: Su Fang , Liu Shu‐Qin , Chen Ying , Chen Fang‐Xia , Wang Hui‐Ping , Xia Qiang
Published: June 2021

Aim

To investigate the effects of luteolin on cardiac functions and mitochondrial oxidative stress in streptozotocin (STZ)‐induced diabetic rats.

Methods

Male Sprague‐Dawley rats were randomly divided into a normal control group, a luteolin control group, a diabetic group, and diabetic groups orally administered with a low dose (10 mg/kg/d) or a high dose of luteolin (100 mg/kg/d) for eight weeks. The body weight, blood glucose, cardiac functions, left ventricular weight, myocardial collagen, and reactive oxygen species (ROS) levels were assayed. The cardiac mitochondrial ROS level, superoxide dismutase (SOD) activity and the mitochondrial swelling were measured.

Results

Treatment with luteolin had no effect on the blood glucose but reduced the losing of body weight in diabetic rats. High dose of luteolin markedly reduced the ratio of ventricular weight and body weight, increased the left ventricular develop pressure, and decreased the left ventricular end diastolic pressure in diabetic rats. The myocardial levels of ROS and collagen, the cardiac mitochondrial ROS level, and the mitochondrial swelling in diabetic rats were all markedly reduced by high dose of luteolin. Furthermore, high dose of luteolin significantly increased the mitochondrial SOD activity in diabetic rat hearts.

Conclusion

Treatment with luteolin for 8 weeks markedly improves the cardiac function, which may be related to reducing mitochondrial oxidative stress and mitochondrial swelling, in diabetic rats.

This work was supported by grants from the National Natural Science Foundation of Zhejiang Province ( Y206179 ).
Mechanical Activation of Smad, A Novel Regulator for Endothelial Cell Proliferation Induced by Disturbed Flow
Authors: Zhou Jing , Lee Pei‐Ling , Lee Chih‐I , Yang Tung‐Lin , Chien Shu , Chiu Jeng‐Jiann
Published: June 2021

Atherosclerotic lesions develop at arterial branches and curvatures, where the local flow is disturbed with low and oscillatory shear stress (OSS). We investigated the role of OSS in the endothelial cell (EC) activations of Smad1/5 and their regulation in OSS‐induced EC proliferation. OSS (0.5±4 dynes/cm2) to human umbilical vein ECs induced sustained phosphorylations of Smad1/5. Smad1/5 activations were found only in the selective patches of ECs overlying foam cell lesions in human coronary arteries. These shear‐activations of Smad1/5 were not inhibited by pre‐treating ECs with Noggin, a specific antagonist of bone morphogenic proteins. Transfecting ECs with specific siRNA of BMPRII, BMPRIB, and αv and β3 integrins inhibited OSS‐induced Smad1/5 phosphorylations. OSS induced sustained increases in the association of αvβ3 integrin with BMPRIB, which was inhibited by BMPRII‐specific siRNA. OSS induced the activations of focal adhesion kinase/Shc/extracellular signal‐regulated kinase, which are required for OSS‐induced Smad1/5 phosphorylations. OSS induced increased expression of cyclin A and decreased expressions of p21CIP1 and p27KIP1 and hence proliferation in ECs. Transfecting ECs with Smad1/5‐specific siRNA inhibited these OSS‐induced responses in ECs. Our findings provide a molecular basis for the role of Smad1/5 in regulating EC proliferation in response to OSS.
Isolation of microvascular endothelial cells from tissues using counterflow elutriation
Authors: James Megan L. , Brock Robert W.
Published: June 2021

The purpose of this study was to optimize the isolation of pure and viable microvascular endothelial cell (EC) populations from a variety of murine tissues. While several methods are reported in the literature, many of these are lengthy and result in low yields. Although counterflow elutriation has been used to isolate a variety of cell types, we are unaware of its use for the isolation of ECs from tissues other than from the liver. Therefore, we proposed to evaluate the conditions necessary for separation of ECs from various tissues. Previously described procedures for isolating liver sinusoidal ECs via counterflow elutriation were optimized using our equipment. Additionally, methods for isolating ECs from aortae and kidney were developed and optimized. All cells were isolated from mice overexpressing endothelial‐specific GFP (Tg(TIE2GFP)287Sato/J). Flow cytometry was used to determine purity of isolated EC populations using staining of GFP, CD31 and DiI(3,3′‐dioctadecylindocarbocyanine)‐labeled acetylated LDL. The methods described herein result in EC populations of at least 98% purity. These cells have been successfully used for downstream applications, including molecular biological assays and cell culture. Counterflow elutriation can be employed to obtain high‐yield, high‐purity EC populations from a variety of murine tissues.

This work was funded, in part, by NHLBI T32 HL090610.
Replicative senescence may be a suitable model for assessing in vivo endothelial cell oxidative stress and inflammation with aging in humans
Authors: Rippe Catarina , Blimline Mark , Nelson Keri , Seals Douglas R
Published: June 2021

Oxidative stress and inflammation develop in the vascular endothelium with aging in rodents and humans and cause dysfunction. In vitro culture of endothelial cells to senescence may be a useful model of endothelial aging in humans, but this has not been established. Human aortic endothelial cells (HAEC) were analyzed by western blotting at early (passage 7–10) and senescent (passage 22–24) stages and compared with endothelial cells obtained in vivo from antecubital veins of healthy young (18–35 yr, n=26) and older (55–71 yr, n=26) adult males and analyzed using quantitative immunofluorescent staining. Nitrotyrosine, a marker of oxidative stress, was increased (P<0.05) with age both in vivo (1.6±0.2‐fold) and in vitro (1.9±0.1‐fold). The oxidant enzyme NADPH oxidase increased with age in vivo (1.6±0.1‐fold, P<0.05) and tended to increase in vitro (1.5±0.1‐fold, P=0.14). The antioxidant enzymes catalase and copper zinc superoxide dismutase (SOD) did not differ with age either in vivo or in vitro, whereas manganese SOD was selectively increased in senescent HAECs (1.6‐fold, P<0.05). The inflammatory proteins tumor necrosis factor α, nuclear factor κ B and monocyte chemoattractant protein‐1 increased (all P<0.05) with age both in vivo (1.6±0.2‐, 1.6±0.2‐, 1.4±0.2‐fold) and in vitro (2.4±0.5‐, 1.7±0.3‐, 1.6±0.1‐fold). In vitro culture of vascular EC to senescence may be a useful model of in vivo EC aging in humans for assessing oxidative stress and protein expression of oxidant and antioxidant enzymes and inflammatory molecules.

NIH AG013038 and the Swedish Medical Research Council
Intra‐arterial adenoviral delivery of GFP‐FRNK inhibits FAK‐ and PYK2 signaling following balloon injury
Authors: Koshman Yevgeniya E , Chu Miensheng , Samarel Allen M
Published: June 2021

The focal adhesion kinase family of nonreceptor protein tyrosine kinases ‐ focal adhesion kinase (FAK) and proline‐rich tyrosine kinase‐2 (PYK2)‐play a central role in integrin‐dependent vascular smooth muscle (VSMC) signaling. FRNK (FAK‐Related Non‐Kinase) the autonomously expressed C‐terminus of FAK, inhibits both FAK and PYK2 signaling in cultured VSMC. To determine the role of FAK, PYK2 and FRNK in vascular remodeling, we subjected rats to carotid artery balloon injury and analyzed their expression by Western Blotting. FAK, FRNK and PYK2 were all upregulated (FAK=3.5±0.56; PYK2=6.0±2.2; FRNK=8.9±2.8‐fold) vs uninjured contralateral control arteries 2wk later. To analyze if early FRNK overexpression inhibits FAK and/or PYK2 dependent signaling, adenoviruses (Adv) expressing GFP or GFP‐FRNK were delivered to the arterial lumen at the time of injury. Tissue extracts 1wk later were analyzed with GFP antibody to detect transgene expression, and FAK and PYK2‐ dependent signaling with phosphospecific antibodies. GFP‐FRNK overexpression inhibited FAK‐Y397 phosphorylation, as well as paxillin phosphorylation at Y118. GFP‐FRNK also downregulated PYK2 expression and its phosphorylation at Y402. To analyze whether specific knockdown of FAK or PYK2 attenuates neointimal formation, 2 specific Adv‐shRNAs were designed to target either FAK or PYK2 and were successfully tested in cultured VSMC. We are now examining their use in vivo to ascertain which kinase is responsible for paxillin phosphorylation and downstream signaling. Supported by the Falk Medical Research Trust.
Caveolin‐1 negatively regulates a metalloprotease‐dependent EGF receptor transactivation by angiotensin II
Authors: Takaguri Akira , Shirai Heigoro , Hinoki Akinari , Rizzo Victor , Eguchi Satoru
Published: June 2021

Objective

The EGF receptor (EGFR) transactivation pathway via an ADAM17‐dependent ligand shedding is the key signaling step by which angiotensin II (AngII) induces vascular smooth muscle cell (VSMC) remodeling. However, the molecular mechanism of the ADAM17 regulation remains largely unclear. We hypothesized that caveolin‐1 (Cav1), the major structural protein of a membrane microdomain, caveolae, is involved in the regulation.

Methods & Results

Phosphorylation and expression of the signaling proteins were evaluated in VSMCs prepared from rat aorta. Infection of adenovirus encoding Cav1 markedly inhibited AngII‐induced EGFR ligand shedding, EGFR transactivation, hypertrophy and migration, but not intracellular Ca2+ elevation. Interestingly, ADAM17 localized in Cav1‐rich lipid raft fractions of VSMCs, and that AngII induced a fraction shift of ADAM 17 from low to high density in the fractions. In addition, methyl‐beta‐cyclodextrin, a reagent to disrupt the caveolae structure, stimulates the EGFR ligand shedding and subsequent EGFR activation in VSMCs.

Conclusion

Caveolae provides an indispensable base environment for activation of AngII signal transduction linked to vascular remodeling. Moreover, our data indicate that dissociation of Cav1 from ADAM17 is a crucial mechanism by which AngII induces ADAM17 activation.
Atorvastatin Attenuates Sox9 Joint and Cardiac Calcification in ApoE null mice
Authors: Park Jeffrey , Flores Amy , Basu Nivedita , Krueger Bryan , Rajamannan Nalini M.
Published: June 2021

Background

Degenerative joint disease is responsible for high morbidity in patients. The mechanisms of osteoarthritis and cardiac calcification are under investigation. We hypothesize that lipids may play a role via chondrogenesis.

Methods

We tested lipids with and without statins in the ApoE mouse to determine the Sox9 expression in the joints and valves. ApoE−/− mice (n=60). Group I (n=20) normal diet, Group II (n=20) 0.25% chol diet (w/w), and Group III (n=20) 0.25% (w/w) chol diet+atorv for the development of abnormal matrix synthesis in the joints over 24 weeks. The aortic valve (AVA) was examined for Bone formation and was assessed by microCT. Aortic Valve Disease was assessed by echo.

Results

The chol diet induced bone in the AV and thickened knee joints with an increase in Sox9 and Cyclin as compared to the Control(>3‐fold(p<05). Atorvastatin attenuated all markers. Echo demonstrated a mild increase in the peak jet velocity. Masson Trichrome demonstrated a marked increase in the matrix synthesis.

Conclusion

We demonstrated that chondrogenic mediated bone formation in the hypercholesterolemic aortic valves and abnormal matrix synthesis in the knee joints is attenuated with atorvastatin.
Flow‐induced mechanotransmission from the endothelial cytoskeleton to extracellular matrix
Authors: Evans Rachel E. , Mott Rosalind E. , Helmke Brian P.
Published: June 2021

Dynamic integrin ligation and transient Rho GTPase inactivation are required for endothelial cell adaptation to hemodynamic shear stress, but bidirectional mechanosignaling mechanisms between extracellular matrix (ECM) and the cytoskeleton remain unknown. In this study, GFP‐vinculin and rhodamine‐fibronectin displacement were tracked simultaneously during a step increase from 0 to 12 dyn/cm2 unidirectional shear stress. In postconfluent monolayers, shear stress onset caused downstream‐directed displacement of focal adhesions and fibronectin fibrils. Transient inactivation of RhoA after shear stress onset was mediated by activation of p190RhoGAP‐A. In subconfluent layers, displacement patterns after shear stress onset in fully assembled ECM were similar to those in confluent monolayers, and cell motility was increased. On partially assembled fibronectin, structural dynamics were arrested after shear stress onset. Overexpression of dominant‐negative p190RhoGAP‐A eliminated flow‐dependent cessation of displacement. These data suggest that shear stress regulates RhoA‐dependent mechanotransmission to the ECM via p190RhoGAP‐A. Supported by NIH grant HL080956.
Life‐long caloric restriction confers pronounced AMPK‐dependent cardioprotection
Authors: Edwards Andrew G , Donato Anthony J , Lesniewski Lisa A , Gioscia Rachel A , Seals Douglas R , Moore Russell L
Published: June 2021

AIMS

To determine whether myocardial resistance to ischemia/reperfusion (I/R) injury is enhanced in the senescent heart after life‐long caloric restriction, and whether AMP‐activated protein kinase (AMPK) plays a role in that phenotype.

METHODS AND RESULTS

Hearts from ad libitum fed (AL) and life‐long calorically restricted (LCR, 40% restriction) B6D2F1 mice were examined by echocardiography, and 25/90 minute global I/R. AMPK inhibition (AraA) was applied to both groups (AL+AraA, LCR+AraA) prior to I/R. LCR hearts were smaller (AL, 270 ± 7 mg vs. LCR, 185 ± 5 mg, p < 0.001), and had thinner posterior walls (PWTd: AL, 1.07 ± 0.05 mm vs. LCR, 0.84 ± 0.05 mm, p < 0.05), but similar in vivo luminal dimension (LVIDd; LVIDs: AL, 3.8 ± 0.14; 2.0 ± 0.19 mm vs. LCR, 3.5 ± 0.18; 1.8 ± 0.17 mm, both p > 0.2). LCR hearts were protected from infarction (Infarct Size: AL, 28 ± 4 % vs. LCR, 10 ± 1 %, p < 0.01) and post‐ischemic functional deficit (LVDP recovery: AL, 65 ± 8 % vs. LCR, 93 ± 7 %, p < 0.01), and both effects were abolished by AraA (Infarct size: LCR+AraA, 22 ± 4 %; LVDP recovery: LCR+AraA, 82 ± 9 %, both p vs. AL > 0.1). LCR dramatically increased AMPKα phosphorylation (AL, 1.30 ± 0.03 vs. LCR, 1.55 ± 0.03, ratio units, p < 0.001), but not expression (AL, 1.21 ± 0.07 vs. LCR, 1.19 ± 0.01, ratio units, p = 0.8).

CONCLUSIONS

Life‐long caloric restriction induces powerful AMPK‐dependent I/R resistance, and involves elevated AMPKα phosphorylation.
Insulin resistance as a mechanism of preserved myocardial function in heart failure animals fed a high fat diet
Authors: Christopher Bridgette A , Huang Hsuan‐Ming , McElfresh Tracy A , Chen Xiaoqin , Muzic Raymond F , Croniger Colleen M , Chandler Margaret P
Published: June 2021

Heart failure (HF) rats fed a high saturated fat diet (SAT) have improved left ventricular contractile function compared to HF animals fed normal chow (NC) diets. Both HF and SAT are individually associated with the development of insulin resistance (IR), but the role of IR in HF+SAT is unknown. We hypothesize that the cardioprotective effect seen in HF+SAT results from defects in insulin signaling that decrease glucose uptake in IR myocardium. Rats underwent coronary ligation or sham (SH) surgery & were fed NC or SAT for 8 weeks. Cardiac contractility (peak positive dP/dt) was preserved in HF+SAT animals versus HF+NC. SH+SAT, HF+NC & HF+SAT animals have abnormal glucose tolerance tests & elevated insulin levels compared to SH+NC, indicating peripheral IR. MicroPET data shows HF+SAT animals have myocardial IR relative to all other groups. Protein targets of insulin signaling (IRS‐1, Akt, pAkt, GLUT4) are currently being analyzed. HF+SAT animals are IR in both peripheral tissue & the myocardium, while SH+SAT & HF+NC animals are IR in the periphery only. This correlates to preserved contractile function in HF+SAT versus HF+NC. We predict that insulin signaling targets such as pAkt and GLUT4 are downregulated, which may be the mechanism by which myocardial insulin resistance preserves contractile function in HF+SAT animals. (Grant # NHLBI HL‐081857)