bluuebunny/crossref_metadata_2025 huggingface dataset:
Apratoxin‐A Induces Pancreatic Toxicity
Authors: Akare Sandeep , Condon Krista , Eckley Sean , Huang Kuan‐Chun , Chen Zhihong , Jiang Yimin , Kotake Yoshihiko , Van Gessel Yvonne , Hutto David
Published: June 2021

Objective of this study was to characterize toxicity induced by Apratoxin‐A, a marine cyanobacterium derived natural product with anti‐cancer activity. In mice, severe weight loss in absence of decreased food consumption or activity was seen, and defined the maximal tolerated dose (MTD). Histopathological evaluation revealed pancreatic atrophy with widespread apoptosis, cell loss and small/collapsed lobules but without cell necrosis, neutrophilic inflammation and fibrosis, and thus mediated by an apoptotic rather than a necrotic mechanism. A tissue bio‐distribution study showed accumulation of Apratoxin‐A in pancreas and mandibular salivary gland at a very high concentration compared to liver, kidney and plasma; however, histologically, the salivary gland acinar cells had decreased cytoplasm but no apoptosis or cell loss. The selective apoptosis and cell loss in pancreas may be due to highly sensitive pro‐apoptotic safeguarding mechanism(s) in the pancreatic exocrine cells to prevent leakage of pancreatic enzymes and avoid severe tissue damage, inflammation and fibrosis. There was no cytotoxicity present in the intestine up to the MTD dose indicating that the Apratoxin‐A activity does not target rapidly dividing cells, but instead acts through functional inhibition of a target protein, likely Sec61, an ER protein important in secretory protein translocation that is expressed at significantly high levels in exocrine glands: pancreas, salivary gland and prostate. In summary, Apratoxin‐A shows selective pancreatic toxicity that is associated with high bio‐distribution to pancreas as well as susceptibility of pancreas for functional inhibition of Apratoxin‐A target protein.
Effects of Cyclic Nucleotide Phosphodiesterase 2 (PDE2) Inhibition on Object Recognition Memory in Mice
Authors: Lueptow Lindsay , Xu Ying , O'Donnell James
Published: June 2021

Cyclic nucleotide phosphodiesterases (PDEs) are a super‐family of enzymes responsible for regulating intracellular levels of the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP). PDE2 is of special interest due to its high level of expression in forebrain regions, which are highly implicated in memory processes. In order to examine the role of PDE2 in memory, we trained mice in the object recognition test (ORT) and administered the PDE2 inhibitor Bay60‐7550 30, 60 or 90 min prior to training (acquisition), immediately after training, 1 hr or 3 hrs after training (consolidation), or 30 min prior to testing (recall). All testing was done 24 hrs after training. When administered via IP injection up to 60 min prior to training or immediately after training, Bay 60‐7550 (3 mg/kg) significantly increased object memory. However, when Bay 60‐7550 was administered 1 or 3 hrs after training, we did not see a change in ORT memory. When given 30 min prior to testing, Bay 60‐7550 had a slight, but non‐significant increase in ORT. We also investigated 3 doses of Bay 60‐7550 (1 mg/kg, 3 mg/kg and 6 mg/kg) at the most robust time point, 30 min prior to training, and saw a significant increase in ORT memory 24 hrs later for all doses. Based on these findings, PDE2 inhibition enhances ORT memory when administered during acquisition and early consolidation memory processes, but not late consolidation or recall. Future research will investigate the precise signaling molecules in the PDE2 pathway that confer these time‐dependent effects on memory.
Effects of Synthetic Cannabinoid JWH‐018 and Phytocannabinoid Δ9‐THC on Learning and Memory in Mice
Authors: Caperton Caitlin , Camper Kiristen , Tai Sherrica , Fantegrossi William
Published: June 2021

The growing popularity of botanical and synthetic cannabinoids among adolescents has resulted in concerns regarding the effects of cannabinoid abuse on certain cognitive processes in the developing brain. Here, the effects of exposure to the phytocannabinoid Δ9‐tetrahydrocannabinol (Δ9‐THC) or the synthetic cannabinoid JWH‐018 on learning and memory were studied in adult mice using two behavioral assays. In passive avoidance studies, mice were placed into a light compartment of a chamber and exposed to three electrical stimulus pulses upon crossing into a dark compartment. The latency to cross was measured prior to conditioning and during subsequent exposures to the chamber. Operant studies included an active‐versus‐inactive nose‐poke discrimination, a nose‐poke contingency reversal, and an extinction condition. In all phases, the number of sessions required to reach criterion performance was measured. Injections of saline, 30 mg/kg Δ9‐THC or 3 mg/kg JWH‐018 were administered in both assays. In passive avoidance studies, chronic treatment with JWH‐018 prior to conditioning significantly reduced crossover latencies, consistent with a profound deficit in learning. In operant studies, neither acquisition nor extinction were affected, but Δ9‐THC treatment dramatically impaired reversal learning. These results suggest that Δ9‐THC or JWH‐018 use may produce deficits in learning depending upon the frequency and patterns of use, and that these two cannabinoids may impact different cognitive domains. These studies were funded by NIGMS IDeA Program award(GM110702), UAMS Translational Research Institute(RR029884) and by ASPET SURF awards to COC and KDC.
Disruption of Normal Sleep Cycles in SULT4A1Δ8/Δ8 Mutant Zebrafish
Authors: Crittenden Frank , Falany Charles
Published: June 2021

For 14 years, sulfotransferase (SULT) 4A1 has presented an enigma to the field of cytosolic SULT biology. SULT4A1 is exclusively expressed in neural tissue, is highly conserved, and has been identified in every vertebrate studied to date. Despite this singular level of conservation, no substrate or function for SULT4A1 has been identified. Previous studies demonstrated that SULT4A1 does not bind the obligate sulfate donor, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), yet SULT4A1 is classified as a SULT superfamily member based on sequence and structural similarities to the other SULTs. In this study, transcription activator‐like effector nucleases (TALENs) were used to generate heritable mutations in the SULT4A1 gene of zebrafish. The mutation (SULT4A1Δ8) consists of an 8 nucleotide deletion within the second exon of the gene, resulting in a frameshift mutation and premature stop codon after 182 aa. During early adulthood, casual observations were made that mutant zebrafish were exhibiting sleep‐like behaviors during the day inconsistent with published reports on sleep in zebrafish. Zebrafish are largely diurnal organisms whose sleep is markedly inhibited by light. Thus, an increase in sleep‐like behavior during the day represents an abnormal behavior and warranted further systematic analysis. Using EthoVision video tracking software, this study demonstrates a significant increase in total sleep time and sleep bout frequency during the day in SULT4A1Δ8/Δ8 fish. Night time sleep patterns remained unchanged. The identification of a physiological process (sleep), the normal function of which depends upon expression of SULT4A1, represents a major step forward in the search for this orphan SULT's function. (Supported by NIH grant R21MHO95946)
RGS2 Protein Degradation is Mediated by a Novel Cullin 4B/F‐box 44 E3 Ligase Complex
Authors: Sjogren Benita , Storaska Andrew , Swaney Steven , Neubig Richard
Published: June 2021

Hypertension and heart failure are major health issues and there is a need to identify more effective treatments. Regulator of G protein Signaling 2 (RGS2) is highly expressed in both heart and vascular smooth muscle and regulates Gαq signaling. RGS2‐/‐ mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Over‐expression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease.

RGS2 is rapidly degraded through the proteasome, but the specific enzymes involved are not known. Thus, the goal of the current study was to identify the molecular machinery responsible for RGS2 protein degradation. A siRNA screen to identify genes that regulate RGS2 protein levels identified components of a putative cullin‐RING E3 ligase (CRL). siRNA knock‐down of F‐box 44 (FBXO44) or cullin 4B (CUL4B), but not CUL4A or CUL1, led to a significant increase in RGS2 protein levels and stability. Overexpression of FBXO44 and CUL4B decreased RGS2 protein levels. FBXO44 associates with both RGS2, CUL4B and the adaptor protein DDB‐1 in cells resulting in a novel E3 ligase protein complex able to degrade RGS2. Studies are now underway to characterize the molecular mechanisms of the FBXO44‐RGS2 interaction.

RGS proteins are difficult drug targets due to their mode of action being through protein‐protein interactions. Inhibiting a more druggable protein within the degradation pathway could be a way to increase RGS2 protein levels and function.
Towards the Identification of Small Molecule Inhibitors of the GIV‐Gi Interaction as Potential Anti‐metastatic Drugs
Authors: DiGiacomo Vincent , Nguyen Lien , Blanco‐Canosa Juan , Garcia‐Marcos Mikel
Published: June 2021

Trimeric G proteins are classically activated by G protein‐coupled receptors (GPCRs). Dysregulation of this mechanism contributes to myriad pathologies from cancer to psychiatric disorders. Indeed, GPCRs are the target for >30% of marketed drugs. G proteins can also be activated by non‐receptor proteins, however, representing a novel signaling mechanism for which no pharmacological modulators exist. Among these newly identified regulators, the GIV protein is a particularly attractive pharmacological target owing to its tight association with cancer metastasis. Moreover, GIV‐Gi coupling underlies the molecular basis of metastasis. The available structural and biochemical information indicates the interface is a tractable target for small molecules. Here, we report the development of assays compatible with high throughput screening (HTS) to monitor the direct interaction between GIV and the Gαi3 G protein. We mapped the minimal Gαi3‐binding sequence in GIV and used it to develop a fluorescence polarization‐based assay that performed robustly in a 384‐well format (Z’ ~0.7). We also developed an orthogonal counterscreen (AlphaScreen) compatible with HTS (384‐well, Z’ ~0.8). A pilot screen of the LOPAC1280 library identified 2 validated hits. Based on literature searches and in silico docking, we provide further evidence that these 2 molecules can directly target the GIV‐Gαi interface. Taken together, these results indicate that the GIV‐Gαi interface is a “druggable” protein‐protein interaction and set the framework for identifying potent anti‐metastatic agents.

Support: NIH R01GM112631
Molecular Mechanisms of Phospholipase Cβ Regulation
Authors: Lyon Angeline , Begley Jessica , Manett Taylor , Tesmer John
Published: June 2021

Phospholipase C β (PLCβ) enzymes hydrolyze phosphatidylinositol‐4,5‐bisphosphate to produce the second messengers inositol‐1,4,5‐triphosphate and diacylglycerol. PLCβ enzymes are autoinhibited by an X—Y linker region within catalytic core that occludes the active site and a helix from within the C‐terminal extension. Protease protection assays, site‐directed mutagenesis, biochemical assays, and X‐ray crystallography were used to investigate the molecular basis of autoinhibition by these regulatory elements and define their respective roles. Distinct regions within the X—Y linker were shown to have specific roles in regulating access to the PLCβ active site, with the conserved acidic stretch within the linker being the most autoinhibitory element. Crystal structures of PLCβ3 variants lacking the acidic stretch have disordered X—Y linkers and bind IP3. The C‐terminal inhibitory helix acts independently of the linker and prevents full activation of the enzyme in the absence of Gαq. Our results demonstrate that PLCβ activation is a multi‐step process requiring independent molecular events induced by membrane association and the binding of activating proteins.
Proteomic Approach to Understanding the Mechanism of Action of New Pharmaceutical Molecules That Impinge on the Eukaryotic Translational Control
Authors: Peker Selen , Ozel Demiralp Duygu , Aktas Bertal
Published: June 2021

Regulation of translation initiation through eIF2 alpha (eIF2a) phosphorylation plays a critical role in cellular homeostasis. eIF2 cycles between GTP‐bound active and GDP‐bound inactive forms. Phosphorylation of eIF2a on Ser51 blocks eIF2B catalyzed GDP‐GTP exchange, reduce the dissociation of eIF2 from eIF2B and inhibits global mRNA translation. Molecular modifiers and effectors of translational control by eIF2a phosphorylation are not fully understood. Global proteomic studies may be useful to elucidate molecular effectors and modifiers of eIF2a phopshorylation using diarylurea compound, BTdCPU, that induces eIF2a phosphorylation by activating heme regulated kinase. We utilized transgenic human prostate cancer (PC‐3) cell lines in which endogenous eIF2a is replaced by either a nonphosphorylatable eIF2a mutant (S51A) or a recombinant wild‐type eIF2a (WT) to identify differentially expressed proteins by treating PC‐3 cells (WT or S51A) with active (BTdCPU)or inactive compound. Two dimensional electrophoresis (2DE) was performed for each group and changes in protein expression in response to BTdCPU treatment were compared by PDQuest software.From 363 differentially expressed protein spots,56 were found to be statistically significant and were further analyzed by MALDI‐TOF MS using Mascot PMF analysis.17 proteins were identified from 12 differentially expressed protein spots. Data will be presented to show specific pathways activated or inhibited by eIF2a phosphorylation and their role in cellular processes.
Neuroprotective effect of flavonoids, via up‐regulating Nrf2‐ARE pathway, in MPP+‐induced PC12 cells, as a model of Parkinson's disease
Authors: Kulkarni Priyanka , Benzeroual Kenza
Published: June 2021

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by intra neuronal proteinaceous cytoplasmic inclusions known as Lewy Bodies. A line of evidence reported formation of dopamine quinones; mitochondrial dysfunction and glutathione loss in SN leads to formation of reactive oxygen species and oxidative stress which ultimately leads to dopaminergic cell death associated with PD. Studies have suggested that flavonoids exhibit a variety of biological activities. However, most interest has been devoted to the antioxidant activity of flavonoids, which is due to their ability to reduce free radical formation and to scavenge free radicals. This study investigated cytoprotective effects of Fisetin and Quercetin against MPP+ induced oxidative stress and neurodegenration in PC12 cells involving NF‐E2‐related factor‐2 (Nrf2) mediated up‐regulation of the antioxidant enzyme NAD(P)H quinone oxidoreducase‐1 (NQO1), Heme Oxygenase‐1(HO‐1) and glutathione. Our results demonstrated that pretreatment with Fisetin and Quercetin not only protected the PC12 cells from MPP+ induced toxicity by increasing cell viability, but also significantly induced the expression of Nrf2‐induced cytoprotective genes NQO‐1, HO‐1 and glutathione. This study emphasized a link between the formations of reactive oxygen species due to mitochondrial dysfunction, depletion of glutathione and dopamine oxidation and also suggests cytoprotective mechanism offered by both Fisetin and Quercetin. This study is supported by research funds of Arnold & Mary Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn NY.
Identification of MicroRNAs Involved in the Regulation of Human UGT1A, UGT2B7 and UGT2B15 Gene Expression
Authors: Papageorgiou Ioannis , Court Michael
Published: June 2021

Background

The human UGT enzymes are responsible for the glucuronidation and elimination of a large number of clinically important drugs. Large interindividual variations of human hepatic drug glucuronidation have been observed in vitro, which cannot be entirely explained by the mechanisms studied so far. MicroRNAs (miRs) are small, non‐coding RNAs that regulate gene expression in a sequence‐specific manner. At present little is known about their role in the regulation of human UGTs. Here we sought to identify miRs that bind to the 3′UTR sequences of UGT1A (identical in all functional UGT1A isoforms), UGT2B7, and UGT2B15, representing the majority of UGTs involved in human drug glucuronidation.

Methods

HEK293 cells were co‐transfected with a plasmid carrying the Firefly luciferase gene under the control of the respective UGT 3′UTR (wild‐type allele), a plasmid with Renilla luciferase (normalizer) and a miR. Using this approach we screened a commercial library containing all known human miRs.

Results

The analysis identified multiple miRs (6 each for UGT1A and 2B7; 16 for 2B15) that down‐regulated reporter activity. Deletion of the predicted miR response element (MRE) abrogated this down‐regulation. Interestingly two miR‐21‐3p MREs in the UGT1A 3′UTR contained two different SNPs (one in each) that did not affect miR binding. Finally UGT1A and 2B15 shared common MREs for miR‐376b‐3p and miR‐103b.

Conclusions

We have identified 28 unique miRs that are likely involved in the regulation of expression of the majority of UGTs responsible for drug glucuronidation in humans. Further experiments will verify the effect of these miRs on UGT gene expression and enzyme activity in human cell lines and primary cultures.
Central Pathways of MAPKp38 and MAPKJNK Mediate TNFα/iNOS‐Dependent Endotoxic Manifestations of Hypotension and Compromised Heart Rate Variability in Rats
Authors: Sallam Marwa , El‐Gowilly Sahar , Abdel‐Galil Abdel‐Galil , El‐Mas Mahmoud
Published: June 2021

We investigated whether TNFα‐coupled mitogen‐activated protein kinases (MAPKs) in central neurons modulate the early hemodynamic responses evoked by endotoxemia in rats. Changes in blood pressure (BP), heart rate (HR), and HR variability (HRV) caused by i.v. lipopolysaccharide (LPS, 10 mg/kg) were assessed in absence and presence of pharmacologic inhibitors of TNFα, inducible nitric oxide synthase (iNOS), MAPKERK1/2, MAPKp38, or MAPKJNK. Compared with saline, LPS caused significant falls in BP and increases in HR that appeared 30 min post treatment and continued for the following 60 min. The time (standard deviation of beat‐to‐beat intervals, SDNN, and the root mean square of successive beat‐to‐beat differences in R‐R intervals, rMSSD) and frequency domain indices (total power and spectral bands of low and high‐frequency) of HRV were all reduced by LPS. The inhibition of TNFα (pentoxifylline) or iNOS (aminoguanidine) abolished the hemodynamic and HRV effects of LPS. Intracisternal (i.c.) injection of ODQ (guanylate cyclase inhibitor), wortmannin (PI3K inhibitor), SP600125 (MAPKJNK inhibitor) mitigated the LPS‐evoked reductions in BP and HR, but failed to affect the associated decreases in HRV. MAPKp38 inhibition by i.c. SB203580 produced exactly the opposite effects. The reduction in BP or HRV caused by LPS remained unaltered after i.c. PD98059, suggesting no role for MAPKERK1/2 in the LPS responses. Together, the data suggest that central TNFα/iNOS/GC/PI3K pathways linked to MAPKp38 and MAPKJNK are pivotal for provoking the reductions in BP and HRV, respectively, seen during endotoxic shock.
Effect of Pentoxifylline on the hypertension in insulin resistance: Role of low‐grade inflammation
Authors: Azhar Ahmad , El‐Nassossy Hany
Published: June 2021

Objectives

The present study investigated the protective effect of pentoxifylline (PTX) from hypertension in IR.

Methods

MetS was induced in rats by high fructose (10% in drinking water) high fat diet for 12 weeks to develop hypertension. PTX was daily administered (30 mg.kg‐1) in the last 4 weeks of study before the blood pressure (BP) parameters were fully investigated in anaesthetized rats via a microtip catheter inserted in the aorta via an opening in the carotid artery. In addition, serum levels of glucose, fructosamine, insulin, tumor necrosis factor alpha (TNFα), adiponectin and lipid profile were determined.

Results

Feeding the rats with high fructose high fat diet led to development of IR as evidenced by the significant hyperinsulinemia and hyperglycemia while PTX administration did not affected the developed IR. IR animals were characterized by significant increase in the mean BP while PTX administration completely prevented the IR associated elevations in mean BP. PTX prevented the elevations in systolic, diastolic and notch BP in IR animals. However, PTX did not affect the elevations in pulse BP seen in IR animals. Neither IR nor PTX affected the heart rate or ejection or non‐ejection durations. In addition, PTX alleviated the low grade inflammation associated with IR as reflected by the significant decrease in serum TNFα and increase in serum adiponectin levels compared with IR animals.

Conclusions

PTX directly alleviates hypertension in IR via amelioration of peripheral resistance and the low grade inflammation
Role of Krüppel Like Factor (Klf4) in Pulmonary Fibrosis
Authors: Perkins Tina , Wary Kishore
Published: June 2021

Rationale

The mature endothelial cells (ECs) encounter with and must respond to several ‘intrinsic’ and ‘extrinsic’ endothelial disrupting factors and inflammatory cytokines. In this regard, KLF4 has been suggested to play an EC protective role; however, the underlying mechanisms are poorly understood.

Goal

To address the role of Klf4 in adult lung microvessels by deleting EC‐Klf4 in a timed manner.

Methods and Results

Immunostaining showed significant reduction of KLF4 expression in human emphysema lung tissue samples. Cigarette smoke condensate decreased expression of KLF4 in ECs. Therefore, to address the role EC‐Klf4 in mice, male Klf4fl/+ were crossed with female Klf4fl/+ mice to generate Klf4fl/fl (25%)offspring. Next, Klf4fl/fl mice were crossed with male tg‐Cdh5CreERT2 mice to produce heterozygous Klf4fl::tg‐Cdh5CreERT2 offspring (100%). Thereafter, male Klf4fl::tg‐Cdh5CreERT2 were crossed with female Klf4fl::tg‐Cdh5CreERT2 to produce Klf4fl/fl::tg‐Cdh5CreERT2 siblings (25%). Groups of these mice received tamoxifen (TAM) everyday for 5 days, and on the 10th day (called Klf4ECKO mice) were sacrificed. Accordingly, TAM treatment decreased EC‐Klf4‐protein expression significantly in the Klf4ECKO lug compared to Klf4fl/fl ortg‐Cdh5CreERT2 controls. Klf4ECKO lung tissue showed increased recruitment of neutrophils and inflammatory cells. Trichrome staining revealed increased collagen deposition in the Klf4ECKO versus wild‐type mice. Importantly, the number of lung alveoli decreased (p<0.05 vs control, n=5), hemorrhage in Klf4ECKO mice compared to the control. Accordingly, the number of CD31+ vascular structures decreased in these mice.

Klf4 protect ECs from ‘intrinsic’ and ‘extrinsic’ endothelial disrupting factors, therefore its loss results in pulmonary fibrosis.
ER‐α Receptors Have More Prominent Depressor Role On Vasoconstractile Sensitivity in Treated And Untreated‐Ovariectomized Rat Mesentery Artery
Authors: Akhayeva Tamila , Ozansoy Gulgun
Published: June 2021

Natural estrogens have cardioprotective effects in premenopausal women. Nuclear estrogen receptors mediate genomic effects of estrogens. G‐protein coupled estrogen receptors are also defined cardiovascular system and mediate rapid non‐genomic effects. The role of estrogen receptor ER‐α and ER‐β on vasculature system is not completely understood. In this study, we investigated the effects of ER‐α and β on α1‐adrenergic receptor mediated vasoconstraction in control (C), 16‐week ovariectomized (O) and 17‐β estradiol (E2) treatment ovariectomized‐rat (OE) mesentery arteries. Phe‐mediated concentration response curves (CRC) were obtained from C, O and OE groups in the presence and absence of non‐selective ER agonist E2 (0.1μM), selective ER‐α agonist PPT (0.1μM) and selective ER‐β agonist DPN (0.1μM). Phe‐mediated CRC shifted to right presence of PPT in C (pD2:6.32 vs pD2: 5.80), O (pD2:6.40 vs pD2: 5.79) and OE (pD2: 6.52 vs pD2:6.08) groups. After E2 incubation, Phe‐mediated CRC only shifted to right in C group (pD2:6.40; pD2: 5.80), but insignificantly shifted to right in O (pD2:6.40 vs pD2:6.13) and OE (6.52 vs. 6.21) groups. On the other hand, DPN incubation did not change Phe‐mediated CRC in all groups.

In conclusion, ER‐α receptor mediated responsiveness have more prominent depressor effects on vasocontractile sensitivity in all group.
Identification of a Prostate Cancer Cell Proteinase Activated Receptor/MMP Signaling Axis
Authors: HOLLENBERG Morley , Mihara Koichiro , Liu Yu , Ramachandran Rithwik
Published: June 2021

Hypothesis

Proteinase Activated Receptors (PARs) are G‐Protein‐Coupled Receptors activated by the proteolytic unmasking of a cryptic tethered ligand. We proposed that prostate cancer cells produce PAR‐regulating enzymes.

Methods

We monitored PC3, DU145 and LNCaP cell supernatant cleavage of PARs in intact cells by fluorescence imaging using N‐ and C‐ terminus dual‐fluorophore‐tagged PARs. Loss of the N‐terminal tag denotes receptor cleavage‐activation. We also used N‐terminal nano‐luciferase‐tagged PARs that, via luciferase release, document PAR‐cleaving proteinase activity in cell‐derived supernatants. MAPKinase and transwell migration assays were used to monitor PAR activation in PC3 cells.

Results

Activation of PARs 1 & 2 triggered MAPKinase signaling and cancer cell migration. Distinct PAR‐cleaving proteinases in conditioned media from all three prostate cancer cell lines were observed with the N‐Luc‐Luciferase assay, with differential inhibition by matrix metalloproteinase inhibitors and soya trypsin inhibitor. Further, when expressed in PC3 cells, the dual‐fluorophore‐tagged PAR1 was seen in a constitutively cleaved‐active state, with an absent N‐terminus.

Conclusion

Our data show that prostate cancer‐derived cells secrete PAR‐regulating proteinases that, via an autocrine process, can regulate PAR function in the tumour microenvironment. Since PAR activation triggers prostate cell migration and MAPKinase, PAR antagonists and inhibitors of PAR‐activating proteinases represent therapeutic targets for treating prostate cancer.

Funding

Prostate Cancer Canada Movember Discovery grant.
Age‐Related Decline of Autocrine Pituitary Adenylate Cyclase‐Activating Polypeptide Impairs Angiogenic Capacity of Rat Cerebromicrovascular Endothelial Cells
Authors: Banki Eszter , Sosnowska Danuta , Tucsek Zsuzsanna , Gautam Tripti , Toth Peter , Tarantini Stefano , Tamas Andrea , Helyes Zsuzsanna , Reglodi Dora , Sonntag William , Csiszar Anna , Ungvari Zoltan
Published: June 2021

Aging impairs angiogenic capacity of cerebromicrovascular endothelial cells (CMVECs) promoting microvascular rarefaction, but the underlying mechanisms remain elusive. PACAP is an evolutionarily conserved neuropeptide secreted by endothelial cells and neurons conferring important antiaging effects. We hypothesized that age‐related changes in autocrine PACAP signaling contributes to impaired angiogenic capacity. Primary CMVECs were isolated from young (3 mo) and aged (24 mo) F344xBN rats. Expression of PACAP was decreased in aged cells associated with impaired capacity to form capillary‐like structures, adhesiveness to collagen (ECIS technology), and increased apoptosis (caspase3). PACAP overexpression in aged cells resulted in increased capillary‐like structure formation. Treatment with recombinant PACAP significantly increased endothelial tube formation and inhibited apoptosis in aged CMVECs. In young cells shRNA knockdown of PACAP expression impaired tube formation, mimicking the aging phenotype. Cellular (DHE) and mitochondrial (MitoSox) ROS production increased in aged CMVECs and were unaffected by PACAP. Collectively, PACAP exerts proangiogenic effects and age‐related dysregulation of PACAP signaling may contribute to impaired angiogenic capacity of aged CMVECs.
PMN Depletion Alters Ang‐1:Ang‐2 & Increases Tie2 Phosphorylation Following Hemorrhagic Shock Priming for the Development of iARDS in Mice
Authors: Lomas‐Neira Joanne , Chung Chun‐Shiang , Ayala Alfred , Chen Yaping
Published: June 2021

Successful therapies for acute respiratory distress syndrome(ARDS) remain challenging. We have recently shown, in our mouse model of hemorrhage priming (Hem) for the development of ARDS following subsequent septic challenge, that depletion of peripheral blood neutrophils(PMN), prior to Hem, reduces lung inflammation/tissue injury and alters ratio of endothelial cell(EC) growth factors, Angiopoietin(Ang)‐1&2. Ang1&2 modulate EC activation, competing for a shared receptor, Tie2, constitutively expressed on ECs. Ang‐1, released by pericytes, promotes an anti‐inflammatory/pro‐survival and Tie2 phosphorylation, while Ang‐2, stored preformed and released from activated ECs, promotes decreased barrier function/increased lung permeability and inhibits pTie2. In PMN depleted ARDS mice, plasma and lung tissue Ang‐2 is significantly decreased. To understand how this relates to reduced lung tissue injury we assessed changes in phopho‐(p)Tie2 vs. total Tie2 expression in lung tissue from PMN depleted mice, undepleted Controls as well as Naive mice and those receiving an Ang‐2 blocking protein. While no statistical change in total Tie2 protein was measured in any of the four groups following ARDS, pTie2 was significantly decreased in lung tissue from mice with PMN (Controls) compared with PMN depleted mice and mice given Ang‐2 blocking protein. pTie2 in lungs of naïve mice was between these 2 groups. This data suggests that while no significant change in Tie2 receptor expression was associated with ARDS in mice, EC/PMN interaction, in addition to altering lung tissue Ang‐2:Ang‐1 ratio, increases pTie2 levels, promoting increased barrier function and decreased lung tissue injury.(NIH GM103652)
Characterization of Intracellular Ca2+ Handling in Vascular Smooth Muscle Cells Isolated from Amputated Human Limbs
Authors: Bruning Rebecca , Sturek Michael
Published: June 2021

High Ca2+ scores assessed by computed tomography in the lower limbs of patients with peripheral artery disease are associated with a greater risk of amputation and all‐cause mortality. Direct assessment of intracellular Ca2+ handling in peripheral vascular smooth muscle cells (VSM) of humans likely to undergo an amputation is unknown due to the invasiveness of such measures. We directly characterized intracellular VSM Ca2+ handling in arteries from amputated human limbs stratified by the number of diagnosed cardiovascular (CV) risk factors (<2 vs >6 CV risk factors). VSM were enzymatically isolated from peripheral arteries and were digitally imaged using the fluorescent intracellular Ca2+ indicator, fura‐2. Cells were exposed to 80 mM K+ in physiologic Ca2+ to assess Ca2+ entry through voltage‐gated Ca2+ channels (VGCC). Caffeine was used in the presence and absence of extracellular Ca2+ to release intracellular Ca2+ from the sarcoplasmic reticulum (SR). VSM from patients with >6 CV risk factors had elevated basal cytosolic Ca2+ (0.76±0.01 vs 0.72±0.01 F360/F380) and greater Ca2+ entry through VGCC (0.08±0.01 vs 0.05±0.00 ΔF360/F380) vs patients with <2 CV risk factors. In the absence of extracellular Ca2+, the SR Ca2+ store release was decreased in VSM in patients with >6 CV risk factors (0.16±0.02 vs 0.22±0.01 F360/F380). However, this decrease was absent in the presence of extracellular Ca2+, suggesting elevated Ca2+‐activated Ca2+ influx with more CV risk factors. Taken together, these data suggest that changes in VGCC, SR Ca2+ stores, and Ca2+‐activated Ca2+ influx act to elevate cytosolic Ca2+ in VSM of amputees with more CV risk factors. NIH T32 HL079995, Health‐IUSM Strategic Research Initiative
Cross‐talk of Leptin and TSP‐1 in Leptin‐induced Atherosclerosis
Authors: Sahu Soumyadip , Ganguly Rituparna , Raman Priya
Published: June 2021

Hyperleptinemia is an important pathogenic contributor of atherosclerosis; however, underlying mechanisms are poorly understood. We reported earlier that leptin upregulates a potent proatherogenic protein, thrombospondin‐1 (TSP‐1), in human aortic smooth muscle cells (HASMC) in vitro and aortic vessels of C57BL/6J mice in vivo. The goal of the current study was to determine the molecular mechanisms by which leptin upregulates TSP‐1 and further delineate the role of TSP‐1 in leptin‐induced atherosclerosis. Using ChIP, we found that leptin in vitro enhanced recruitment of nuclear proteins IRF1 and CREB on TSP‐1 gene promoter in HASMC. Immunoblotting further revealed that leptin in vivo, at doses reported to increase TSP‐1, stimulated IRF1 expression in aortic lysates of WT mice vs. untreated controls; in contrast, leptin failed to increase IRF1 expression in aortic vessels of TSP‐1‐/‐ mice. Finally, to elucidate a direct role of TSP‐1 in leptin‐induced atherosclerosis, age‐matched ApoE‐/‐ and TSP‐1‐/‐/ApoE‐/‐ mice on regular chow at 17 wks of age were treated once daily i.p. with leptin (5µg/g) for 3 wks. While leptin‐treated TSP‐1‐/‐/ApoE‐/‐ had lower plasma cholesterol levels compared to leptin‐treated ApoE‐/‐ mice, FPLC fractionation of plasma lipids indicated that this difference was not attributable to VLDL and LDL levels. Morphometry of aortic root demonstrated a significant increase in atherosclerotic lesion area, collagen content and PCNA expression (proliferation marker) in ApoE‐/‐ vs. TSP‐1‐/‐/ApoE‐/‐ mice in response to leptin, suggesting that TSP‐1 deletion protects against leptin‐induced atherosclerosis. Overall, our data suggest a potential cross‐talk between leptin and TSP‐1 that may play a critical role in hyperleptinemia‐induced atherosclerosis.
GLP‐1 and its Analogue Liraglutide Affects Human Endothelial Cell Signaling by Reducing VEGF‐A Induced Ca2+ Signaling and Activating Protein Kinases
Authors: Rotboel Cecilie , Hansen Anker , Tritsaris Katerina , Xu Jin , Aalkjær Christian , Dissing Steen
Published: June 2021

GLP‐1 is a gut hormone and has been in focus for its role in response to food intake. An analogue of GLP‐1 has been constructed by adding a palmitoyl chain to the GLP‐1 peptide making it adhere to albumin. Here we have explored the potential roles of GLP‐1 and Liraglutide in vascular function by studying signaling in human dermal endothelial cells (HDMEC), human umbilical vein endothelial cells (HUVEC) and rat mesenteric resistance arteries. We found that HDMEC expresses the GLP‐1R by use of RT‐PCR. By use of Ca2+ imaging we found that preincubation with GLP‐1 (500 nM) caused a reduction in VEGF‐A (25 ng/ml) induced Ca2+ signaling due to a reduction in phosphorylation of PLCγ and that GLP‐1 causes phosphorylation of Src, Akt, ERK 1/2 and p38. In HUVEC, VEGF‐A (3 h, 20 ng/ml) causes a 2 times upregulation of mRNA for I‐CAM which is attenuated by 20% in the presence of Liraglutide (100 nM) and a 5 times upregulation of mRNA for V‐CAM which is attenuated 25% by Liraglutid. Liraglutide, but not GLP‐1, caused a small transient increase in [Ca2+]i. Our data are consistent with the GLP‐1 signaling being evoked through an enhanced Gs/βγ signaling since Gi inhibition with pertussis toxin elicits a similar response by favoring the Gs/βγ signaling pathway. The attenuating responses on VEGF‐induced Ca2+ transients and VEGF‐induced mRNA expression for surface proteins implicate that GLP‐1 signaling could affect endothelial cell function in relation to inflammatory processes.
Acute Effects of an External Pneumatic Compression Device on Local and Systemic Resistance Vessel Reactivity and Limb Blood Flow
Authors: Martin Jeffrey , Borges Alexandra
Published: June 2021

High pressure external pneumatic compression (e.g. EECP) has been shown to have marked benefits on vascular reactivity and biology. However it is expensive, uncomfortable, and not easily accessible. Alternatively, low pressure EPC is commercially available, does not require medical supervision, and is low risk.

Purpose

The purpose of this study was to determine the acute effects of low pressure EPC on resting limb blood flow and resistance vessel reactivity in compressed and non‐compressed limbs of healthy young subjects.

Methods

Ten (n=10) apparently healthy subjects (aged 27.0 ± 10.2 years) were enrolled in this randomized, crossover design study. Subjects reported in the morning, fasted, for two visits separated by at least 24‐hours. Prior to and immediately following application of EPC or sham for 60 minutes, forearm (FBF) and calf blood flow (CBF) were evaluated at baseline and during 3 minutes of reactive hyperemia (RH) using venous occlusion plethysmography. A peristaltic pulse dynamic compression device was employed for EPC treatment with target inflation pressures of ~70 mmHg applied to the lower limbs.

Results

EPC significantly improved peak CBF, but no differences in resting FBF, peak FBF, resting CBF, total CBF and total FBF were observed.

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Conclusions

EPC significantly improves acute resistance vascular reactivity in compressed limbs, but not non‐compressed limbs, following 60 minutes of treatment. Therefore, application of low pressure EPC may be efficacious in situations where local alterations of resistance vessel reactivity is desirable.

Partial support for this study was provided by the Quinnipiac University Interdisciplinary Program for Research & Scholarship and by NormaTec (Newton Centre, MA)
Drospirenone Potentiates the Endothelium Dependent Vasodilation in Coronary Bed of Hypertensive Ovariectomized Rats
Authors: Claudio Erick , Borgo Mariana , Silva Fabricio , Romero Walckiria , Gouvêa Sônia , Moyses Margareth , Santos Roger , Almeida Simone , Podratz Priscila , Graceli Jones , Abreu Glaucia
Published: June 2021

This study investigated the effects of combined therapy with E2 and drospirenone (DRSP) on the endothelium‐dependent vasodilation of the coronary bed of ovariectomized spontaneously hypertensive rats. Animals were randomly divided into Sham operated (Sham), ovariectomized (OVX), ovariectomized treated with E2 (E2) and ovariectomized treated with E2 and DRSP (DRSP) groups. Hemodynamic parameters were evaluated by cannulation of the femoral artery. Coronary endothelium‐dependent vasodilation was assessed using isolated hearts. Protein expression of eNOS and estrogen receptor alpha (ER‐α) were assessed by Western blotting. Morphometric parameters were analyzed by histology, and oxidative stress was assessed in situ by dihydroethidium fluorescence. Ovariectomy enhanced systolic blood pressure, which was prevented only by the DRSP treatment. OVX causes endothelial dysfunction, an effect prevented by both treatments; however, the vasodilator response in the DRSP group was potentiated at the three highest concentrations compared to the OVX group. ER‐α expression decreased in OVX rats and was restored by the treatments. Morphometric parameters and oxidative stress were augmented by OVX and reduced by both the E2 and DRSP treatments. Hormonal therapy with E2 and DRSP can be established as an therapeutic option in the prevention of coronary artery disease in hypertensive post‐menopausal women.
The interaction between fluid wall shear stress and solid circumferential strain affects endothelial gene expression
Authors: Amaya Ronny , Tarbell John
Published: June 2021

Endothelial cells lining the walls of blood vessels are exposed simultaneously to wall shear stress (WSS) and circumferential stress (CS) that can be characterized by the temporal phase angle between WSS and CS (stress phase angle – SPA). Regions of the circulation with highly asynchronous hemodynamics (SPA close to ‐180°) such as coronary arteries are associated with the development of pathological conditions such as atherosclerosis and intimal hyperplasia whereas more synchronous regions (SPA closer to 0⁰) are spared of disease. The present study evaluates endothelial cell gene expression of 42 atherosclerosis‐related genes under asynchronous hemodynamics (SPA=‐180 °) and synchronous hemodynamics (SPA=0 °). This study used a novel bioreactor to investigate the cellular response of bovine aortic endothelial cells (BAECS) exposed to a combination of pulsatile WSS and CS at SPA=0 or SPA=‐180. Using a PCR array of 42 genes, we determined that BAECS exposed to non‐reversing sinusoidal WSS (10±10 dyne/cm2) and CS (4 ± 4 %) over a 7 hour testing period displayed 17 genes that were up regulated by SPA = ‐180 ⁰ , most of them pro‐atherogenic, including NFκB and other NFκB target genes. The up reglation of NFκB by SPA =‐180o was confirmed by Western blots and immunofluorescence staining demonstrating the nuclear translocation of NFκB. These data suggest that asynchronous hemodynamics (SPA=‐180 °) can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA may be an important parameter characterizing arterial susceptibility to disease.
Increased Regional Pannexin1 (Px1) Channel Expression linked to Focal Ischemia is Pro‐Arrhythmic During Stress
Authors: Huke Sabine , Faria Thais , Bennuri Sirish , Chepurko Elena , Gumina Richard , Knollmann Bjorn
Published: June 2021

Mice with the hypertrophic cardiomyopathy‐linked TnT‐I79N mutation develop arrhythmias during physiological stress. In isolated hearts these arrhythmias are linked to regional ischemia (no decrease in global coronary flow) which causes regional hypoxia and cellular energy deprivation and results in regional conduction velocity slowing and increased arrhythmia susceptibility (84.7% vs 14.3% in control).

To test if regional hypoxia/ischemia also occurred spontaneously in vivo in TnT‐I79N mice, we measured mRNA expression of genes up‐regulated by hypoxia (GLUT1, TUBB3, Px1) and found increased expression (0.3fold, 3fold, 10fold respectively). To determine the spatial distribution of Px1‐hypoxia marker we first subjected Px1 ‐βGal reporter mice to coronary ligation and demonstrate that Xgal staining is limited to the perfusion bed of the ligated artery. Then we crossed the TnT‐I79N with the Px1 reporter mice and found that Xgal staining increased in a spatially non‐uniform “patchy” distribution, consistent with focal ischemia. Px1 channels have been implicated in the regulation of vascular perfusion during hypoxia. Strikingly, ablation of Px1 in TnT‐I79N mice reduced the arrhythmia incidence in isolated hearts (to 36.4%) and prevented the focal energy deprivation during stress.

Although the physiological role for the increase in Px1 after ischemia remains unclear, Px1 contributes to the increased arrhythmia susceptibility in TnT‐I79N mice. Hence, our data suggest that Px1 may be involved in the regulation of local coronary perfusion and is maladaptive during stress.
Increased Endothelial Expression of Human Soluble Epoxide Hydrolase Negatively Modulates Coronary Reactive Hyperemia in Isolated Mouse Heart
Authors: Hanif Ahmad , Zeldin Darryl , Nayeem Mohammed
Published: June 2021

Coronary reactive hyperemia (RH) is impaired in hypertrophic hearts. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia / reperfusion injury. Soluble epoxide hydrolase (sEH) is the main enzyme responsible for EETs breakdown. EETs level is decreased in transgenic mice with increased endothelial expression of sEH (Tie 2‐sEH tr). We hypothesized that the isolated mouse heart with increased expression of sEH (Tie2‐sEH Tr) exhibits decreased RH through increased hydrolysis of EETs in response to no‐flow ischemia compared to wild type (WT). Coronary flow (CF) in isolated Tie2‐sEH Tr and WT mouse heart was measured using Langendorff system. Perfused isolated heart was exposed to 15 second ischemia and RH was assessed. Following ischemia, flow repayment volume (RV) (the area under the curve and normalized to heart weight (ml/g)) was significantly decreased in Tie2‐sEH Tr compared to WT(8.7 ± 0.8 in Tie2‐sEH Tr vs. 10.7 ± 0.7 in WT, p<0.05). Moreover, Tie2‐sEH Tr exhibited significantly shorter repayment duration when compared to WT (2.4 ± 0.2 in Tie2‐sEH Tr vs. 3.3 ± 0.4 in WT, p<0.05). At the same time, left ventricular developing pressure (LVDP) and heart rate (HR) were not significantly different between the two groups (LVDP was 168.0 ± 15.1 in Tie2‐sEH Tr vs. 133.1 ± 11.6 in WT, p>0.05; HR was 397.7 ± 24.5 in Tie2‐sEH Tr vs. 387.0 ± 14.1 in WT, p>0.05)

Our results demonstrate that increased endothelial expression of sEH (Tie2‐sEH Tr) decreases RH compared to WT. These data suggest that increased level of sEH could have a deleterious effect on myocardial recovery from ischemia. Supported by HL‐114559 to MAN and z01‐ES025034 to DCZ
Effects of Ageing on Nitric Oxide (NO)‐Mediated Cerebrovascular Tone and Autoregulation
Authors: Coney Andrew , Thompson Emma , Marshall Janice
Published: June 2021

Vascular tone in cerebral circulation is partly determined by NO‐mediated dilatation. NO has also been implicated in the cerebral vasodilatation in response to hypercapnia/hypoxia and at the lower limit of autoregulation. However the role of NO at the upper limit (UL) is unclear. Further, as ageing leads to impaired endothelium‐dependent vasodilatation, we hypothesised these roles of NO may diminish.

In anaesthetised male Wistar rats, non‐selective NOS inhibition by L‐NAME (10mg/kg i.v.) increased arterial blood pressure (ABP) by 20±2 and 26±6mmHg in young (Y: 6‐8 weeks, n=16) and old (O: 52‐58 weeks, n=7) rats respectively; concomitantly, gross cerebral blood flow (CBF) recorded from common carotid with external ligated, fell by 0.7±0.1 and 1.2±0.3ml/min.

We then infused phenylephrine (0.1–200µg/kg/min i.v.) to raise ABP. The autoregulatory UL for gross CBF was calculated by dual‐line linear‐regression of CBF vs ABP. Similarly, cortical red cell flux (cRCF) was monitored by laser speckle imaging and cRCF UL was calculated. In Y rats after L‐NAME, gross CBF UL was either 187±2mmHg (n=4) or not reached even when ABP was maximally increased to 186±3 (n=12). cRCF UL was also not reached when ABP was increased to 187±2mmHg (n=8). These values are higher than gross CBF UL generated in Y control rats without L‐NAME (168±2mmHg*; P<0.05). In O rats after L‐NAME, neither gross CBF nor cRCF UL was reached in any animal, even though ABP reached 199±5mmHg§ (§: vs Y after L‐NAME). This was also higher than gross CBF UL in O control rats (180±3mmHg*).

We suggest NO induces tonic cerebral dilatation in both Y and O animals, such that inhibition allows greater scope for autoregulatory vasoconstriction, raising the UL.

British Heart Foundation funding is gratefully acknowledged.