bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatocyte growth factor activator inhibitor type 1 (HAI‐1) is required for the integrity of intestinal epithelium
Authors: Kawaguchi Makiko , Fukushima Tsuyoshi , Takeda Naoki , Hoshiko Shinri , Yorita Kenji , Kohama Kazuyo , Orikawa Hiroshi , Baba Takashi , Kataoka Hiroaki
Published: June 2021

Hepatocyte growth factor activator inhibitor type 1 (HAI‐1) is a membrane‐bound serine protease inhibitor expressed in placenta and epithelial tissues. HAI‐1 inhibits several serine proteases such as hepatocyte growth factor activator, matriptase, prostasin and hepsin. The hai‐1 knockout (KO) mice were embryonic lethal due to impaired formation of placental labyrinthine. When we rescued placental function, the KO mice were viable but died by 16 days after delivery with ichthyosis‐like skin abnormalities. In this study, we generated intestinal epithelial cell‐specific hai‐1 KO mice, as hai‐1 is most abundant in the intestinal epithelium. These mice developed normally in appearance; however, they exhibited histological abnormalities of crypts in part of the colon, showing decreased goblet cells. They also showed increased intestinal permeability. To study the role of hai‐1 in inflammation, we induced colitis with 1% dextran sodium sulfate (DSS) in drinking water for 4 days followed by administration of distilled water without DSS. After DSS treatment, KO mice had more severe symptoms as indicated by loss of body weight, diarrhea and bloody stool. Moreover, survival rate was lower than control mice. Autopsy revealed enhanced mucosal edema and erosion particularly in the cecum. These results suggest that HAI‐1 plays an important role in maintaining the integrity of intestinal epithelium.
Diversity and activation of rhesus Paneth cell α‐defensins
Authors: Llenado R. Alan , Maemoto Atsuo , Shanahan Michael Thomas , Ouellette Andre J.
Published: June 2021

Paneth cells (PCs) occur in small intestinal crypts from which they secrete α‐defensins at high levels. To investigate rhesus macaque enteric α‐defensin (REDs) diversity, ileal mucosal protein and mRNAs were characterized. Analyses of ileum α‐defensin cDNA and gene sequences identified 20 distinct RED cDNAs and 7 RED genes not represented in the cDNA population. Certain deduced RED proteins coded by abundant mRNAs have mutations at canonical α‐defensin structural features. α‐Defensin proteins isolated from ileum occur predominantly as proREDs with negligible processed REDs detected by acid‐urea PAGE and MALDI‐TOF MS. In vitro, purified natural and recombinant proREDs were activated by trypsin to yield structurally intact, mature RED peptides. Because mature RED peptides are lacking from ileal PCs and mouse PC α‐defensins persist in colonic lumen (J. Biol. Chem. 284: 27848–27856, 2009), colonic luminal contents were assayed for evidence of post‐secretory proRED processing. Mature, intact RED peptides were evident in large bowel lumen, far downstream from their small intestinal site of secretion, and they were identified as α‐defensins by Edman sequencing. The processed state of α‐defensins in rhesus PCs resembles that in human PCs and differs from the mouse PCs, where activated, mature α‐defensins are abundant in PC dense core granules. Supported by NIH grants AI059346 and DK044632.
Cell phones as a new m‐Health opportunity for mass cervical cancer screening
Authors: Markovic Olivera , Markovic Nenad
Published: June 2021

Lacking an efficient preventive screening, cervical cancer is a problem in developing world taking one woman's life every three minutes. At the prior EB meetings, we presented our MarkPap® platform technology for early detection of abnormal cells on cervical specimens, subsequently upgraded with telecytopathology using digital imaging and web networking. We will present expanding this platform into m‐Health using a modified cellphone camera technology for capturing microscopic images and their wireless transmission for distant evaluation.

The biomarker is a unique connecting link between the microscopic evaluation on site and a remote, digital image evaluation by experts at distant locations. One of the still unresolved challenges to enable wider application of this wireless technology is the positioning and keeping the camera sensor in a proper illumination position to the ocular plane. We are currently testing several cellphone adapters to develop a universal adapter to connect “any microscope” and “any cellphone camera.” Comparing images taken with standard digital camera and the cellphone camera with our new adapters we have shown sufficient cytological details enabling cytological diagnosis with high inter‐observer reliability.

These results indicate this tool (m‐Health) as a new affordable prospective for cervical cancer screening and for saving women's lives in the low‐resource areas.
Epac1 is Up‐regulated in Chronic Lymphocytic Leukemic B‐Cells
Authors: Brown Loren , Zhang Lingzhi , Kipps Thomas , Insel Paul
Published: June 2021

Exchange protein activated by cAMP 1 (Epac1) is a guanine‐nucleotide exchange factor (GEF) for the small G‐protein Rap and is involved in proliferation and anti‐apoptosis. Epac1 has recently been recognized as a key mediator of cAMP actions. Limited previous studies have noted that Epac1 expression is up‐regulated in chronic lymphocytic leukemia (CLL). To investigate the up‐regulation of Epac1 in CLL, we assessed peripheral blood mononuclear cells (PBMC) and found that Epac1 mRNA (determined by real‐time PCR) and protein (by immunoblotting) are up‐regulated ~400 fold and ~10 fold, respectively, in CLL patients compared to controls (even if we control for enrichment of B cells). We hypothesize that the increase in Epac1 expression is due to increased transcriptional activity of the Epac1 gene. To test this hypothesis, we cloned the 2kb promoter region directly upstream of the start site for Epac1 into the pGL4.10 luciferase reporter vector. Deletional analysis revealed that the region necessary for basal expression of Epac1 is located within 100 base pairs of the transcriptional start site. These results indicate that Epac1 is up‐regulated in PBMC of CLL patients and that this is not attributable to the enrichment of B‐cells. As the up‐regulation of Epac1 is unique to the CLL B‐cell, determining its mechanism should provide insight into the biology of CLL.
VEGFxxxb is relatively down‐regulated in malignant neuroblastoma but not in benign ganglioneuroma
Authors: Peiris Maria , Bates Dave , Ramani Pramila
Published: June 2021

Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and is up‐regulated by variety of tumours. An endogenous family of anti‐angiogenic isoforms termed VEGFxxxb has been identified in many normal, non‐angiogenic tissues, and, in contrast with the angiogenic VEGFxxx isoforms, is down‐regulated in epithelial tumours including colorectal and prostate carcinoma. This is the first study of the anti‐angiogenic variant VEGF165b in neuroblastic tumours of childhood, which range from highly malignant neuroblastoma to benign ganglioneuroma.

Twenty neuroblastic tumour samples were assessed for VEGFxxx and VEGFxxxb mRNA and protein expression by RT‐PCR and ELISA, respectively. VEGFxxx but not VEGFxxxb was up‐regulated in neuroblastoma (N=12) when compared with ganglioneuroma (N=6) at mRNA level. At the protein level, the ratio between VEGFxxxb and total VEGF was significantly lower in neuroblastomas (0.53±0.08; N=15) than in ganglioneuromas (0.96±0.23; N=4). Immunohistochemistry identified VEGF165b in ganglioneuroma but not neuroblastoma, which showed higher expression of total VEGF. These results suggest that alternative splicing of VEGF in neuroblastoma may contribute to its malignant characteristics. Research funded by the CLIC Sargent
P2 Receptors and ATP Enhance the Migration of Prostate Cancer Cells
Authors: Peters Wachen , Sikes Robert A , Duncan Randall , Maguire Christine
Published: June 2021

Purinergic signaling stimulates many biological processes. Two classes of purinergic receptors, GPCR (P2Y) and gated channels (P2X), have been identified that bind ATP as a ligand, which can promote cell migration, a critical component of metastasis. ATP also has been shown to have an anticancer effect in vivo. The purpose of this study was to ascertain the effect of ATP on the migration of an isogenic progression series of prostate cancer (PCa) cell lines on three different extracellular matrices. We hypothesize that ATP treatment activates purinergic receptors that increases cell migration on all three extracellular matrices (ECM).

RT‐PCR was used to determine the mRNA expression profile of P2 receptors and ecto‐nucleotidases. Migration was examined using wound healing assays as followed by photo microscopy for four days, as well as RT‐PCR to determine effect of interaction with each ECM on the mRNA profile. ATP increased the migration of LNCaP cells on Collagen I to a higher degree than on both Schwann and matrigel over vehicle alone. Peak wound closure occurred after 48 hours. Differential expression of P2 receptors and ectonucleotidases was seen when cells were grown on different matrices.

ATP is necessary for PCa migration on collagen I, matrigel and Schwann cell matrix. The differential expression of P2 receptors is speculated to be responsible for variable wound healing on extracellular matrices.
Lysophosphatidic acid enhances VEGF‐C expression in PC‐3 human prostate cancer cells
Authors: Lin Chuan‐en , Lin Chih‐Hsin , Chang Chi‐Lun , Huang Yuan‐Li , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid (LPA) is a low‐molecular‐weight lipid growth factor binds to G‐protein‐coupled receptors and activates a variety of physiological functions. VEGF‐C has been shown to regulate the development and growth of the lymphatic system. Our previous studies demonstrated that LPA enhances VEGF‐C in human endothelial cells, suggesting that LPA is an important regulator of lymphatic vessel formation and lymphangiogenesis. However, the roles of LPA on VEGF‐C expression on cancer cells were not elucidated. In this study, we showed that LPA up‐regulated VEGF‐C mRNA and protein expression in PC‐3 human prostate cancer cells. By using chemical inhibitors, we showed that these enhancement effects were LPA1‐ and LPA3‐dependent and required activation of Gi. To further confirm the roles of LPA on VEGF‐C expression in prostate cancer, PC‐3 cells stably expressed shRNA of LPA1 and LPA3 were generated. By real‐time PCR, we showed that the serum‐enhanced VEGF‐C expression is down‐regulated in these cell lines. Our results suggested that LPA regulates VEGF‐C expression in PC‐3 cells. This study provides some basic information that LPA might be a novel target for therapeutics against lymphangiogenesis and tumor metastasis.
Renin/prorenin receptor decreased expression in heart during pregnancy‐induced hypertension in rats
Authors: Lopez‐Sanchez Pedro , Lopez Emilio Moctezuma , Avila Massiel Alfonsina , Anguiano‐Robledo Liliana
Published: June 2021

Pregnancy courses with low vasopressor response to angiotensin II (Ang II). In preeclampsia or pregnancy‐induced hypertension (PIH), this effect is reverted. Recently, it has been reported a novel receptor in the renin angiotensin system, the Renin/Prorenin receptor (RPR). It is not well described the probable role of this receptor during preeclampsia. The aim of this work was to study if RPR modifies its expression in heart during PIH. We used a subrenal aorctic coarctation (SRAC) model in female Wistar rats, measuring blood pressure, proteinuria and pups weight. Heart was weighted and auricular, septum and ventricular tissue samples were homogenized and immunoblotted using an Anti‐RPR antibody. We found hypertension and proteinuria in SRAC‐pregnant rats and RPR expression in both, pregnant non‐SRAC and pregnant SRAC groups. In pregnant non‐SRAC rats, RPR is abundantly detected. In pregnant SRAC rats however, we found a lower expression of this receptor. These results suggest that RPR is expressed in normal pregnancy and decreased expression during preeclampsia may indicate a role in the pathophysiology of pregnancy‐induced hypertension. Supported by SIP‐IPN 20090153 and 20090396 grants
Expression of renin/prorenin receptor is decreased in vessels and kidney during pregnancy‐induced hypertension in rats
Authors: Esteban Rosa , Anguiano‐Robledo Liliana , Vela Jose Alfredo , Lopez‐Sanchez Pedro
Published: June 2021

Pregnancy courses with low vasopressor response to angiotensin II (Ang II). In preeclampsia or pregnancy‐induced hypertension (PIH), this effect is reverted. Recently, it has been reported a novel receptor in the system, the Renin/Prorenin receptor (RPR). It is not well described the probable role of this receptor during preeclampsia. The aim of this work was to study if RPR modifies its expression during PIH. We used a subrenal aorctic coarctation (SRAC) model in female Wistar rats, measuring blood pressure, proteinuria and pups weight. Aortic and renal tissue samples were homogenized and immunoblotted using an Anti‐RPR antibody. We found hypertension and proteinuria in SRAC‐pregnant rats and RPR expression in both, pregnant non‐SRAC and pregnant SRAC groups. In pregnant non‐SRAC rats, RPR is abundantly detected. In pregnant SRAC rats however, we found a lower expression. These results suggest that RPR is expressed in normal pregnancy and decreased expression during preeclampsia may indicate a role in the pathophysiology of pregnancy‐induced hypertension. Supported by SIP‐IPN 20090396 and 20090153
Increased catecholamine content and release from adrenal chromaffin cells of DOCA‐salt hypertensive rats
Authors: Fhaner Matthew J , Swain Greg , Galligan James J
Published: June 2021

Sympathetic neuroeffector transmission to mesenteric arteries is disrupted in DOCA‐salt hypertension but the molecular mechanisms responsible are unknown. Due to their small size, sympathetic nerve terminals are inaccessible to analysis by electrophysiological approaches. Therefore, we used secretory adrenal chromaffin cells as model for sympathetic nerve terminals. These cells store and release catecholamines, norepinephrine and epinephrine. HPLC analysis revealed higher catecholamine content in adrenal gland of DOCA‐salt compared to control rats. Amperometry with microelectrodes was used to measure catecholamine release evoked by acetylcholine stimulation of individual chromaffin cells. Catecholamine release were measured as an oxidation current. Amperometric recordings show in increase in frequency and number of release events per stimulus and quantal amount per event, along with a decrease in rise time and the half‐width for individual events. Results indicate that adrenal tissue stores more catecholamines in DOCA‐salt hypertension. Catecholamines are released from vesicles at a higher frequency and with a larger quantal content.

(Supported by HL70687)
Influence of ACE gene polymorphisms on antihypertensive efficacy, left ventricular mass and proteinuria in children undergoing ramipril monotherapy
Authors: Buescher Rainer , Finkelberg Ilja , Hoyer Peter F.
Published: June 2021

OBJECTIVE

It is unclear whether ACE gene polymorphisms influence the therapeutic outcome of a ACE‐inhibitor therapy. We therefore investigated the role of the ACE I/D polymorphism on 24‐hour blood pressure (ABPM), left ventricular mass index (LVMI) an proteinuria in children following ramipril monotherapy.

METHODS

Sixty children (age 14.4±3.8 years) with various chronic nephropathies and hypertension received ramipril monotherapy (mean maximum dose 5.3±1.6 mg/m2). Their ACE I/D genotype was correlated to results of the 24‐h ABPM, LVMI and amount of proteinuria before and 6 months after treatment.

RESULTS

Mean SDS blood pressure (BP) significantly dropped within 6 months (2.2±1.7 vs. 0.4±1.1 after 6 months, P<0.0001). This effect remains when patients are stratified according to the ACE II or DD genotype (mean SDS‐BP; II: 3.3±0.5 vs. 1.2±0.3, P=0.019; DD: 2.3±0.5 vs. 0.3±0.4, P=0.013). LVMI did not significantly differ before and following initiation of ramipril treatment and a stratification by ACE genotype did not reveal a difference either. However, proteinuria remained significantly higher in patients with the DD genotype.

CONCLUSIONS

Ramipril is an efficacious antihypertensive therapy in children. Whereas the ACE genotype does not have an impact on the LVMI development, the DD‐allele seems to be negatively associated with the antiproteinuric effect of ramipril.
Differential Alteration of Sympathetic Norepinephrine Transporter (NET) in Mesenteric Arteries and Veins in DOCA‐salt hypertensive rats
Authors: Sangsiri Sutheera , Dong Hua , Swain Gregory M , Galligan James J , Xu Hui
Published: June 2021

Increased sympathetic nervous system activity contributes to DOCA‐salt hypertension in rats. Sympathetic nerves release NE that acts on the postjunctional α1 receptors causing vasoconstriction. NE is cleared by NET. We tested the hypothesis that NET function is impaired in DOCA‐salt rats. Video microscopy and amperometry were used to measure neurogenic vasoconstriction and real time NE release in mesenteric arteries (MA) and veins (MV). Neurogenic constriction was larger in DOCA‐salt MA compared to control MA but vascular reactivity to NE was similar in DOCA‐salt and control MA. Cocaine, a NET blocker, increased NE peak current in arteries but not veins. Cocaine caused less increase in NE current in DOCA‐salt MA artery compared to control. Guanethidine caused concentration dependent (10–300 nM) inhibition of NE currents release in control MA, MV and DOCA‐salt MV, but not DOCA‐salt MA. These data suggest NET function is more prominent in MA compared to MV. Increased NE currents in DOCA‐salt MA are due partly to impaired NET function. (Supported by HL70687)
Nitric Oxide Synthase And Soluble Guanylyl Cyclase Activation Are Required For Hypoxic Endothelium‐Dependent Contractions Of The Porcine Coronary Artery
Authors: Chan Calvin KY , Mak Judith CW , Man Ricky YK , Vanhoutte Paul M
Published: June 2021

The vasoconstrictor response to hypoxia in the lung, termed hypoxic pulmonary vasoconstriction, is also observed in coronary arteries. The present study examined the mechanism underlying hypoxic contractions in isolated porcine coronary arteries. Isometric tension was measured in rings with or without endothelium. In quiescent preparations, the contractile response to hypoxia was only observed in rings with endothelium and was abolished by indomethacin and S18886, demonstrating the involvement of cyclooxygenase products and TP receptor activation, respectively. In contracted preparations, the hypoxic response was also endothelium‐dependent and was reduced by indomethacin and S18886. The endothelium‐dependent hypoxic contractions were abolished by L‐NAME, ODQ and NS2028 in both quiescent and contracted preparations. The addition of DetaNONOate in the presence of L‐NAME restored the hypoxic response, suggesting the involvement of the nitric oxide pathway. Assay of the cyclic GMP content showed no change upon exposure to hypoxia in preparations with and without endothelium. The relaxation to 8‐Br‐cGMP was not reduced during hypoxia, meaning that the hypoxic contraction is not due to abolition of a basal relaxing component. These experiments suggest that hypoxic endothelium‐dependent contractions of the porcine coronary artery depend on more than one signaling pathway.
Regulation of Cerebral Blood Flow by Hydrogen Sulfide
Authors: Ahmed Ganiat , Oyekan Adebayo O , Yakubu Momoh A
Published: June 2021

H2S is formed from L‐Cysteine by cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE) and has been implicated in the regulation of neuronal function. Though, CBS is highly expressed in the brain, contribution of H2S to the regulation of cerebral blood flow (CBF) is not well understood. We examined the effects of H2S on CBF in anesthetized rats treated with H2S donor NaHS; precursor L‐Cysteine (50; 20 mg/kg) alone or following inhibition of CBS/CSE with Propargylyglycine (PPG; 75 mg/kg). Changes in CBF were determined by Laser Doppler Scanner (Moor LDI 5152). NaHS or L‐Cysteine increased basal CBF by 19 or 10%, while PPG reduced CBF by 33% (p<0.05). L‐Cysteine had no effect on PPG‐induced reduction of CBF but NaHS elicited a short‐lived reversal (13% at 30 min) that reverted to PPG level 60 min later. H2S‐induced regulation of CBF was influenced by age; L‐Cysteine increased CBF in the young (6 months) (17%) compared to sustained reduction (18%) in the adult (18 months). NaHS elicited a transient (5–15 min) increase in CBF in the young (22%) but produced a sustained reduction in the adult, reducing CBF to 49%. This study shows that the donor, precursor, and inhibitor of H2S synthase modulate CBF with differential effects in the adult versus young rats. Both H2S donor and precursor reduced adult CBF with the reduction being more pronounced by the donor. The differential effects could be due to age‐related loss of CBS/CSE. Thus, H2S synthetic pathway could play a significant role in neuropathology and serve as a therapeutic target for neurological diseases.
Exaggerated vasoconstriction depends on ω‐hydroxyylase through adenosine A1receptor (AR) in A1 WT compared to A1 KO mouse aorta
Authors: Kunduri Swati Sarada , Nayeem Mohammad , Falck John , Schnermann Jurgen , Mustafa S.Jamal
Published: June 2021

Adenosine exerts its vasoconstrictor effect with the activation of A1AR through PLC‐βIII → PKCα → p42/44 pathway, and possibly arachidonic acid derived metabolite (20‐HETE) generated through hydroxylation via CYP4A enzyme in vascular smooth muscle cells. 20‐HETE plays an important role in the regulation of vascular tone in a number of vascular beds. We hypothesize that the A1AR‐mediated vasoconstriction depends on CYP4A hydroxylation of arachidonic acid. Organ bath experiments with aorta from A1WT and A1KO mice were used in this study. The tissues were contracted with 10−6M PE and the intactness of endothelium was tested with 10−6M ACh. Tissues that relaxed >40% were used in this study. A1KO had significantly higher ACh relaxation (+72.38±14.07%) than A1WT (52.76±11.87%). CRC to a non‐selective adenosine analog (NECA) and an A1AR selective agonist (CCPA, 10−6 M) were obtained with CYP4A inhibitors (HET‐0016, 10−6 and DDMS, 10−5M). HET‐0016 changed the NECA (−19.51 ± 3.68%) and CCPA‐induced contraction (−34.08± 5.68%) into relaxation (60.36± 13.7 %). DDMS changed CCPA (10−6M) induced contraction (−29.83 ± 7.19%) into relaxation (18.79± 5.19%). CYP expoxygenase inhibitor (MSPPOH, 10−5M) caused significantly higher contraction in the treated group (−50.46± 6.25%) vs the control (−20.46± 4.82%). These data support a role for CYP4A in A1AR‐mediated vasoconstriction. Supported by HL027339, HL09444, GM31278
A novel signaling pathway for high output eNOS‐derived nitric oxide in inflamed endothelial cells
Authors: Lowry Jessica Leigh , Brovkovych Viktor , Zhang Yongkang , Skidgel Randal A.
Published: June 2021

Under inflammatory conditions, high output NO is thought to derive primarily from inducible nitric oxide synthase (iNOS), but the role of endothelial NOS (eNOS) is not well studied. In normal endothelium, kinin B2 receptor (B2R) signaling results in Ca2+‐dependent activation of eNOS and transient NO production, but in inflamed endothelium, we found that B2R activation leads to prolonged high output eNOS‐derived NO. Human lung microvascular endothelial cells (HLMVEC) were treated with interleukin‐1β (20 ng/ml) and interferon‐γ (200 U/ml) for 16 h to mimic inflammatory conditions. High output NO stimulated with 100 nM bradykinin (BK) was reduced with eNOS siRNA or cNOS inhibitor, measured directly with a porphyrinic electrode. Cytokine treatment did not affect eNOS protein expression or phosphorylation at Ser1177 or Thr495. In contrast to control HLMVEC, BK‐dependent eNOS activation in cytokine‐treated cells is mediated by Gαi/o and ERK and is Ca2+‐independent as it was inhibited with pertussis toxin (PTx) or MEK inhibitor U0126, but not BAPTA‐AM. BK stimulated ERK activation more robustly in cytokine‐treated HLMVEC and was sensitive to PTx. Thus, cytokine treatment promotes B2R coupling to Gαi/o leading to ERK‐dependent activation of eNOS and high output NO. These findings may provide new therapeutic targets for the treatment of vascular inflammatory disorders. Supported by NIH HL60678 & T32 HL007829‐13.
Nitroglycerin Reduces TNF‐α Toxicity To Endothelial Cells but Compromises the Protective Effects of Propofol
Authors: Liu Hui‐min , Lei Shaoqing , Luo Tao , Xia Zhong‐yuan , Liu Yanan , Leung George P , Vanhoutte Paul M , Irwin Michael G , Xia Zhengyuan
Published: June 2021

Supplementation of nitroglycerin (NTG) may increase cardiomyocyte death during ischemia (Histol Histopathol 2009;24(12):1487–98) and the underlying mechanism is unknown. Vascular endothelial cell apoptosis precedes cardiomyocyte apoptosis during myocardial ischemia‐reperfusion, a situation that is associated with increased tumor necrosis factor‐α (TNF) production. We postulated that NTG may exacerbate TNF induced endothelial cell apoptosis by enhancing nitrative stress. Cultured human umbilical vein endothelial cells were either not treated (control), treated with TNF (40 ng/ml) alone or TNF in the presence of NTG, propofol (an anesthetic that scavenges peroxynitrite), or NTG plus propofol, respectively, for 16 hr. TNF increased endothelial cell apoptosis, accompanied by increased protein expression of nitrotyrosine, a footprint of peroxynitrite and an index of nitrative stress. Both NTG and propofol attenuated TNF‐induced cell apoptosis, however this protective effect diminished when NTG and propofol were used in combination and this was accompanied by increased nitrotyrosine expression and protein kinase C‐β2 phosphorylation at site ser660. NTG may have compromised propofol cellular protection by counteracting its peroxynitrite scavenging capacity.

Supported by a Society of Cardiovascular Anesthesiologists Research Starter Grant and NSFC grants (30700789 and 30872447).
Antihyperglycemic effect of Centratherum anthelminticum, Terminalia chebula and Terminalia bellirica extracts.
Authors: Arya Aditya , Haerian Batoul Sadat , Mohamad Mustafa Ali
Published: June 2021

The type 2 diabetes alters obesity and cardiovascular disease risks. Several plants are used for the treatment of type 2 diabetes in India. This study aimed to ascertain the scientific basis for the selected herbs with the highest anti‐diabetic activity in the management of type 2 diabetes. The blood glucose level and glucose tolerance test were determined in hexane, chloroform and methanol extracts of Centratherum anthelminticum seeds, Terminalia chebula and Terminalia bellirica fruits, before and after oral administration (250 mg/kg body weight) in normal rats model. The methanol extracts of these three plants suppressed the rise in blood glucose in normal rats during a glucose tolerance test significantly (p < 0.05) and among them, Centratherum anthelminticum seeds, methanol extract was more remarkable, confirmed by the study for 28 days in STZ‐nicotinamide induced type II diabetic and normal rat models. Thus the finding of this study suggest that feeding of Centratherum anthelminticum methanolic extract has a potential anti‐diabetic function in the management of type 2 diabetes.
Activation of Aldehyde Dehydrogenase 2 Reduces Nitroglycerin‐Induced Cardiac Damage
Authors: Sun Lihan , Mochly‐Rosen Daria
Published: June 2021

INTRODUCTION

Nitroglycerin (GTN) tolerance remains a critical barrier to its full benefit of vasodilation. Recent evidence suggests that GTN tolerance is due to inactivation of aldehyde dehydrogenase 2 (ALDH2). We previously showed that activation of ALDH2 by its activator, Alda‐1, reduced cardiac damage after myocardial infarction (MI). Since sustained treatment of GTN inactivates ALDH2, patients on chronic GTN regimen may be at risk of increased cardiac injury after MI. The current study aims to determine whether sustained GTN treatment increases cardiac injury after MI and, if so, whether Alda‐1 can protect from GTN‐induced exacerbation of cardiac damage.

METHODS

We used a rat model of MI induced by LAD occlusion. Animals were treated with GTN with or without Alda‐1. Animals were sacrificed at various time points after MI to determine the infarct size and cardiac function. In another study, the effect of Alda‐1 on GTN‐induced inactivation of ALDH2 was determined, in vitro.

RESULTS

Prolonged treatment of GTN reduced ALDH2 activity by 30%, in vivo. GTN caused a two‐fold increase in infarct size and decreased fractional shortening, compared with untreated controls. In contrast, we found that Alda‐1 prevented GTN‐induced ALDH2 inactivation, in vitro. Mechanisms of such inactivation will be discussed. Further, animals given Alda‐1 along with GTN had 40% decrease in infarct size and 15% increase in fractional shortening, indicating that Alda‐1 inhibited GTN‐induced cardiac injury. The data suggested a dilemma that patients under sustained GTN treatment are likely to have more severe cardiac damage after MI. Alda‐1 can be a potential therapeutic to protect these patients from such damage.
Estrogen Protects Female Rats Against The Nicotine‐Induced Attenuation Of Reflex Tachycardia: Role Of Central Estrogen Receptors
Authors: El‐Mas Mahmoud M , El‐gowilly Sahar M , Fouda Mohamed A , Saad Evan I
Published: June 2021

Our preliminary studies showed that nicotine attenuates reflex tachycardia in male and not female rats. Here, the hypothesis was tested that the resistance of female rats to the nicotine effect is estrogen‐dependent. Baroreflex curves relating reflex tachycardic responses to decreases in blood pressure evoked by sodium nitroprusside (1–16 μg/kg) were constructed in proestrus, ovarictomized (OVX), and estrogen (50 μg/kg s.c., 5 days)‐replaced OVX (OVXE2) rats. Slopes of the curves were taken as a measure of baroreflex sensitivity (BRS). Nicotine (100 μg/kg) reduced BRS in OVX rats but not in proestrus or OVXE2 rats. The attenuation of reflex tachycardia by nicotine was also evident in diestrus rats, which exhibit low plasma estrogen, and in OVX rats treated simultaneously with E2 and the estrogen receptor antagonist tamoxifen. Experiments were then extended to determine whether central estrogenic pathways modulate the nicotine‐BRS interaction. Intracisteral (i.c.) treatment of OVX rats with E2 (0.2 μg/rat) abolished the BRS attenuating effect of nicotine. This protective effect of E2 disappeared when OVX rats were pretreated with i.c. ICI 182,780, a selective estrogen receptor antagonist. Together, central pathways of estrogen receptors seem to play integral role in the protection offered by E2 against nicotine‐induced barorecetor dysfunction.
Knockout strategy reveals synergy between alpha‐1‐adrenoceptor subtypes and P2X receptors in nerve induced vascular responses
Authors: McGrath John Christie , Simpson Paul C , Daly Craig John , Methven Laura
Published: June 2021

All 3 alpha‐1‐adrenoceptor (AR) subtypes could contribute to vasoconstriction of resistance arteries to sympathetic stimulation and therefore contribute to autonomic regulation of blood pressure. However, the poor selectivity of antagonist drugs and the complexity of identifying the individual contributions of different receptors preclude analysis. Here, we employ double and triple knockouts of the alpha‐1‐AR to analyse responses to electrical field stimulation of the wire‐myograph‐mounted mouse first order mesenteric arteries. The results show that alpha‐1A‐AR and alpha‐1D‐AR act synergistically, particularly at low frequencies, so that antagonists of either receptor produced a powerful blockade. After elimination of all 3 alpha‐1‐ARs, the residual response was blocked by alpha, beta, methylene ATP. Analysis of the double knockouts using antagonists revealed synergism between P2X and alpha‐1A‐AR but not between P2X and alpha‐1D‐AR. These data reveal powerful synergism between purinergic and noradrenergic responses with a degree of adrenoceptor selectivity, pointing to signalling interactions. Supported by British Heart Foundation (PG/05/140/20094 and FS/04/035).
Hydrogen Peroxide Enhances the Expression of Gi Protein in Aortic Vascular Smooth Muscle Cells : Relationship with Adenylyl Cyclase
Authors: Mbong Nathan , Anand‐Srivastava Madhu
Published: June 2021

Reactive oxygen species (ROS) play a critical role in the pathogenesis of many diseases including hypertension, atherosclerosis, and diabetes. We have recently shown that ROS contribute to enhanced expression of Giα protein in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). The current study was undertaken to investigate the effect of H2O2, an experimental mimicker of oxidative stress, on Giα protein expression and the implication of extracellular signal‐regulated kinase (ERK1/2), and phosphatidylinositol‐3 kinase (PI3K) signaling in H2O2‐induced altered expression of Giα protein. Aortic VSMC were treated with different concentrations of H2O2 (50 μM to 250 μM) for different periods of time (30 min to 4 hr). The protein expression was determined by Western Blotting using specific antibodies. H2O2 enhanced the expression of Giα in a concentration and time‐dependent manner with a maximum increase at 100μM for 1hr, whereas Gsα expression remained unchanged. The enhanced expression of Giα was demonstrated by the increased inhibition of adenylyl cyclase by inhibitory hormones such as angiotensin II, oxotremorine, and C‐ANP4–23. We also found that H2O2 induced the phosphorylation of the serine/threonine kinases ERK1/2, and AKT/PKB (protein kinase B). Moreover, MEK, and PI3K inhibitors restored the H2O2–induced enhanced expression of Giα proteins to control levels, suggesting the implication of these proteins kinases in the enhanced level of Giα. In conclusion, the modulation of Gi protein expression by H2O2 in aortic vascular smooth muscle cells involves the ERK1/2, and PI3K signaling pathways.

This study was supported by the Canadian Institutes of Health Research grant
Angiotensin II‐induced reactive oxygen radical production by NAD (P) H oxidase is mediated by arachidnic acid metabolism via cytochrome P4501B1 in vascular smooth muscle cells
Authors: Song Chi Young , Ghafoor Hafiz U.B. , Yaghini Fariborz A , Fang Xiao R , Malik Kafait U
Published: June 2021

We have previously shown that angiotensin II (Ang II) and arachidonic acid (AA) induced migration, proliferation and hypertrophy of vascular smooth muscle cells (VSMCs) is mediated by reactive oxygen species (ROS) generated via cytochrome P450 (CYP) 1B1 (Hypertension 54:e122, 2009). The purpose of this study was to determine the relationship between CYP1B1, AA and NAD(P)H oxidase in the generation of ROS in rat VSMCs. Ang II (200 nM) and AA (10 μM) increased by 1.4‐ and 1.7‐fold ROS production respectively, as measured by fluorescence spectroscopy using dihydroethidium. Treatment of VSMCs with CYP1B1 inhibitor, 2,4,3′,5′‐tetramethoxy‐stilbene (TMS) (100 nM) or transduction of VSMCs with adenovirus CYP1B1 shRNA but not its scrambled control reduced CYP1B1 activity measured using P450 GLO™ assay kit, and abolished Ang II‐ and AA‐induced ROS production and NAD(P)H oxidase activity measured by lucigenin‐enhanced chemiluminescence. Ang II‐ and AA‐induced NOX1 expression was attenuated by TMS and NAD(P)H oxidase inhibitor apocynin; the latter agent abolished ROS production but did not alter CYP1B1 activity in VSMCs. AA metabolism inhibitor, 5,8,11,14‐eicosatetraynoicacid, abolished Ang II‐ and AA‐induced ROS production and NAD(P)H oxidase activity and expression of NOX1. These data suggest that Ang II‐induced ROS production by NAD(P)H oxidase is mediated via AA release and its metabolism by CYP1B1 in VSMCs.
Lipopolysaccharide‐induced expression of VCAM‐1 in human renal mesangial cells
Authors: Chen Jung‐Tsan , Yang Chuen‐Mao
Published: June 2021

Several factors have been shown to trigger the pathologenesis of renal inflammatory diseases. Vascular cell adhesion molecule‐1 (VCAM‐1) is one kind of adhesion molecule participating in the process of polymorphonuclear cells (PMNs) transmigration, which recruit and adhesion to the renal glomerular mesangial cells. Thus, up‐regulation of VCAM‐1 may play a pivotal role in inflammatory responses of kidney. However, the mechanisms underlying LPS‐induced VCAM‐1 expression in HRMCs remains unknown. Western blot, real‐time PCR, promoter luciferase assay and monocytes adhesion analyses showed that in HRMCs, LPS‐induced VCAM‐1 mRNA and protein expression, and promoter activity was attenuated by the inhibitors of p42/p44 MAPK (U0126), p38 MAPK (SB202190) and NF‐κB (Bay11‐7082). In addition, LPS stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125). Besides, we also investigate that ROS is involved in LPS‐induced VCAM‐1 expression, determined by flow cytometry and H2DCFDA assays. These results suggested that in HRMCs, LPS‐induced VCAM‐1 expression was mediated through phosphorylation of MAPKs and NF‐κB pathways, and contributed to leukocyte/HRMCs interaction and promote inflammatory responses.
TNF‐α induces cytosolic phospholipase A2 expression via MAPKs and NF‐κ B in human pulmonary alveolar epithelial cells
Authors: Hsiao Yu‐Chun , Yang Chuen‐Mao
Published: June 2021

Exposure of human pulmonary alveolar epithelial cells to external insults triggers inflammatory processes in the respiratory system. Elevated levels of proinflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) have been found in the airway fluids, which may induce up‐regulation of cytosolic phospholipase A2 (cPLA2) implicated in the pathogenesis of inflammatory diseases. Here, our study aimed to investigate the mechanisms underlying the intracellular signalings involved in cPLA2 expression induced by TNF‐□in human pulmonary alveolar epithelial cells (HPAEpiCs). Western blotting and RT‐PCR analyses showed that TNF‐α‐induced cPLA2 protein and mRNA expression as well as phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK were attenuated by pretreatment with the inhibitors of MEK1/2 (PD98059), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs of p42, p38, and JNK2. Furthermore, TNF‐α‐induced cPLA2 expression was inhibited by a selective NF‐κB inhibitor (Bay11‐7082), consistent with that TNF‐α stimulated both IκBα degradation and NF‐κB translocation in these cells. TNF‐α‐induced NF‐κB translocation was blocked by pretreatment with Bay11‐7082, but not by PD98059, SB202190, and SP600125. These results suggested that in HPAEpiCs, TNF‐α‐induced cPLA2 expression was independently mediated through the activation of MAPKs and NF‐κB pathways.
Mnk1 is essential to efficient murine T cell IFNgamma release
Authors: Burnette Barry Lee , Monahan Joseph B
Published: June 2021

Mnk1 is a MAPKAP kinase activated by ERK or p38 which appears to be non‐essential for normal growth or development based on the lack of any adverse phenotype in knockout mice. A role for Mnk1 has been implicated in inflammatory responses and tumorigenesis, and therefore we investigated the role of Mnk1 in the release of pro‐inflammatory mediators from T cells. We generated an Mnk1 knockout mouse on a DBA1 background, and isolated wildtype and Mnk1‐deficient splenic T cells by negative selection. T cells were stimulated with anti‐CD3 and anti‐CD28 antibodies and after 16 hours, culture supernatants were assayed by electrochemiluminescent ELISA for murine IFN‐γ, IL‐1β, IL‐2, IL‐4, IL‐5, KC, IL‐10, IL‐12, and TNF‐α. While IL‐4 and IL‐12 production were significantly suppressed in knockout T cells relative to wild type, IFN‐γ release was inhibited by greater than 80% of control. These results suggested that Mnk1 may effect IFN‐γ production from inflammatory T cells. Although IFN‐γ priming of innate effector cells may play an important role in modulating inflammatory responses and can abrogate anti‐inflammatory signaling, it also has pleiotropic effects and may ameliorate or exacerbate autoimmune diseases dependent upon condition or stage. We propose Mnk1 may be a therapeutic target for autoimmune diseases by suppressing IFN‐γ production.
Resveratrol enhances the sensitivity of doxorubicin‐mediated cell proliferation, invasion, and migration in human breast cancer cell lines
Authors: KIM Hyung Sik , Kim Tae Hyung
Published: June 2021

The aims of this study were to investigate the synergistic anticancer effects of resveratrol with doxorubicin against human breast cancer cell lines. In this study, the doxorubicin‐resistance MCF‐7 (MCF‐7/ADR) and MDA‐MB‐231 cells were treated with resveratrol and doxorubicin, alone or in combination, and we examined cell viability, cell cycle, apoptosis, migration and invasion. Treatment with doxorubicin 1 μM for 24 hr had no effect on the cell viability of MCF‐7/ADR and MDA‐MB‐231 cells. However, combination of doxorubicin and resveratrol significantly reduced viability of MCF‐7/ADR and MDA‐MB‐231 cells, respectively. Although MCF‐7/ADR and MDA‐MB‐231 cells are relatively resistant to the doxorubicin, resveratrol improved the doxorubicin‐induced cytotoxicity in both cell lines. Taken together, combination treatment with resveratrol and doxorubicin decreased MCF‐7/ADR and MDA‐MB‐231 cells migration up to 60% and 80%, respectively, as compared to doxorubicin treatment alone. Similar to migration assay, there was significant reduced the number of invaded cells in assay using Matrigel‐coated chambers. To determine the pathway of anti‐invasive effects, the expression levels of genes involved in migration and invasion were examined in both cell lines. The MMP‐2, MMP‐9 and plasminogen activator (u‐PA) expression levels were significantly decreased following combination treatment. In addition, the expression levels of TIMPs and RECK were markedly increased in case of combination treatment in both cell lines. In conclusion, these findings suggest that resveratrol might be used as a synergistic agent in the treatment of doxorubicin‐resistance breast cancer.
cAMP‐dependent pathway(s) directs the B‐Raf MAPK‐Mediated Cytosolic Mislocalization of p27kip‐cyclin D1 in Renal Cancer
Authors: Tham Kim Y , Cohen Jennifer D , Monks Terrence J , Lau Serrine S
Published: June 2021

The loss of tuberin, the tuberous sclerosis‐2 (Tsc‐2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc‐2EK/+), and has been recently observed in human renal cell carcinoma tissue. Signaling associated with the expression and cytoplasmic mislocalization of p27 is relatively unknown in renal tumor development. TGHQ transformed renal epithelial cells derived from Tsc‐2EK/+ rats (QTRRE cells), that form renal tumors in nude mice, display cytoplasmic mislocalization of p27 and cyclin D1, and also exhibit high ERK, B‐Raf and Raf‐1 kinase activity. Dibutyryl cAMP or phosphodiesterase inhibitors ([PIs]; pentoxifylline or theophylline) cause an increase in cytosolic p27/cyclin D1 protein levels in QTRRE cells. Additionally, treatment of QTRRE cells with both PIs revealed concomitant decreases in p‐p27 T187 and in its E3 ubiquitin ligase skp1; proteosome inhibition resulted in increased p27/cyclin D1 protein levels. Inhibition of Raf kinases with either sorafenib or B‐Raf siRNA resulted in MAPK down‐regulation of p27. Moreover, an associated decrease in cyclin D1 was observed following p27 siRNA knockdown in QTRRE cells. Collectively these data suggest that the cAMP/B‐Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27‐cyclin D1 in tuberous sclerosis‐gene regulated‐renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27‐cyclin D1 cytosolic stabilization during renal tumor formation.
Human mast cell degranulation is distinguished from selective secretion of TNF through intracellular calcium, energy and mitochondrial morphology dynamics
Authors: Zhang Bodi , Angelidou Asimenia , Alysandratos Konstantinos‐Dionysios , Tagen Michael , Kempuraj Duraisamy , Asadi Shahrzad , Theoharides Theoharis
Published: June 2021

The mast cells are unique bone‐marrow derived immune cells that can secrete pre‐stored mediators through degranulation, such as triggered by Substance P (SP) or release de novo synthesized cytokines stimulated by lipopolysaccharide (LPS). However, differences in these modes secretion are largely unknown. We examined energy production, calcium homeostasis and mitochondrial morphological dynamics in LAD2 mast cell degranulation and selective secretion. A) Mast cells degranulation triggered by SP (10 μM) had two times higher energy consumption than LPS (10 ng/ml) treated cells and control cells. Mitochondria ATP pump blocker Oligomycin (2 μM) inhibited 68% of TNF secretion due to degranulation, but has little effects on de novo synthesized TNF release. B) Mast cells have increased intracellular calcium level 10 seconds after SP stimulation, while LPS did not induce significant intracellular calcium increase. C) Mitochondrial uncoupling protein 2 (UCP2) absorbs calcium from intracellular region and protects cells from calcium toxicity. UCP2 mRNA expression level enhanced ten times after 7 hours SP stimulation, but was unchanged with LPS. D) Mitochondria morphology has tight connection with its function. We showed mast cells treated with SP (10 μM) had significant mitochondrial fragmentation, which was no appeared in LPS stimulation. The present findings indicate that secretion from human mast cells can occur through distinct pathways. Mitochondria may play key role in regulating mast cell activation.

This work was supported in part by NIH grant R01 AR47652 to TCT.
Tecarfarin: A New Oral Anticoagulant and the Characterization of its In Vitro Metabolism Using Rat, Dog, and Human Hepatocytes
Authors: Lightning Luke , Kumaraswamy Padmapriya , Coleman Renee , Irwin Ian , Palme Monica , Druzgala Pascal
Published: June 2021

Tecarfarin is a novel compound being developed for oral anticoagulation therapy for the prophylaxis and treatment of thromboembolic complications associated with atrial fibrillation and other cardiac disorders. Like warfarin, tecarfarin inhibits vitamin K epoxide reductase and is equipotent to warfarin at inhibiting this enzyme. It is metabolized to a single inactive metabolite, ATI‐5900, by human esterases. We compared the biotransformation of tecarfarin in cryopreserved hepatocytes isolated from male rats, dogs and humans and assessed the formation of ATI‐5900. We also scanned for, but did not quantify, several potential Phase I and Phase II metabolites for which chemical standards were not available. In dog and human hepatocytes, metabolism of tecarfarin was much slower than in rat hepatocytes, with less than 20% converted to ATI‐5900 at the end of 6 hours. We did not detect the formation of Phase II metabolites in any of the hepatocyte preparations. These results were supported by experiments where tecarfarin was incubated with rat, dog and human liver microsomes. It is well known that the metabolism of warfarin is dependent on CYP2C9 which is associated with various polymorphisms and, as a consequence, variable elimination. In contrast, the metabolism of tecarfarin is dependent on the ubiquitous esterase enzyme superfamily and therefore, its elimination should be more predictable in vivo.