bluuebunny/crossref_metadata_2025 huggingface dataset:
GPCRs as novel potential therapeutic targets in cancer
Authors: Insel Paul A , Sriram Krishna , Wiley Shu Zhou , French Randall P , Lowy Andrew M
Published: November 2023

G protein‐coupled receptors (GPCRs) are the most widely targeted class of FDA‐approved therapeutics but in cancer, GPCRs are generally only targeted in endocrine tumors. We hypothesize that GPCRs have much broader importance in cancer and that therapeutics directed at GPCRs may have unappreciated utility in the treatment of a wide variety of cancers. To test this hypothesis, we have used unbiased approaches (Taqman GPCR arrays and RNA‐seq), mining of publicly available datasets (such as The Cancer Genome Atlas, TCGA), and validation studies to assess GPCRs in multiple human tumors, cancer cells and stromal cells (cancer‐associated fibroblasts, CAFs). A major focus has been on pancreatic ductal adenocarcinoma (PDAC) tumors and cells and pancreatic CAFs (PCAFs). We find that PDAC cells and PCAFs express >75 different GPCRs, including many orphan GPCRs (without known physiologic agonists) and that a substantial number of these GPCRs are expressed at much higher levels than in normal precursor cells. A cluster of GPCRs is overexpressed in PDAC tumors. Two of these receptors are orphan GPCRs (Orphan 1 and Orphan A), neither of which is mutated but each is highly expressed in PCAFs and PDAC cells, respectively. Both Orphan 1 and Orphan A have at least 2‐fold higher expression in >90% of PDAC tumors in TCGA compared to that of normal pancreas. Higher expression of orphan A is associated with increased mortality and a worse clinical course and Orphan 1 expression correlates with that of multiple fibrotic genes. Importantly, both Orphan 1 and Orphan A are functional and appear to contribute to the malignant phenotype. For example, siRNA knockdown of Orphan 1 in PCAFs blunts production of profibrotic markers and decreases ability of conditioned media from PCAFs to enhance proliferation of PDAC cells. Together, these and other results suggest that GPCRs represent previously unrecognized contributors to cancers besides endocrine tumors and as such, may be druggable, novel targets for the treatment of cancer, including pancreatic cancer, a tumor for which new therapeutics are a critical, unmet need.

Support or Funding Information

National Institutes of Health, National Cancer Institute (CA189477, CA121938, CA155620).
The Cellular Mechanism of Bitter Taste Receptor Mediated Relaxation of Rat Aorta
Authors: Xin Wenkuan , Chen Qian
Published: November 2023

Background

Bitter taste receptors, TAS2Rs, are a family of G‐protein‐coupled receptors. TAS2Rs are expressed in mucous epithelial cells of the tongue which mediate the bitter taste and can sense the potential toxins in the food. Recent studies demonstrate that these receptors are expressed in cells outside the gustatory system as well, such as several of smooth muscle tissues. The activation of TAS2Rs can increase the intracellular inositol triphosphate (IP3) thereby the activity of IP3 receptors (IP3R) on the sarcoplasmic reticulum. The Ca2+ that is released through IP3Rs, subsequently activates the large conductance Ca2+‐ and voltage‐activated K+ (BK) channels, and the activation of BK channels leads to the relaxation of smooth muscle.

Methods

We investigated the expression of TAS2Rs in rat aorta using real time qPCR, the functional effect of TAS2Rs activation on rat aortic contractility using isometric tension recordings, and the underlying cellular signaling pathways in smooth muscle cells using confocal microscopy.

Results

qPCR detected the mRNA for TAS2R107 in aorta. The TAS2R agonist, denatonium, relaxed the aorta pre‐contracted with 60 mM K+ in a concentration dependent manner with an EC50 of 215.2 μM (n=6, N=6). The addition of denatonium, as low as 10 μM, caused a transient relaxation of aorta ring pre‐contracted with L‐phynlephrine (10 μM), and the following accumulative addition of denatonium caused a concentration‐dependent further relaxation (n=6, N=6). Denatonium (500 μM) caused a transient increase of electrical field stimulation‐induced (25 Hz‐5 volts) aorta contractions followed by a complete inhibition (n=6, N=6). The confocal microscopy experiments with smooth muscle tissue suggest that the activation of TAS2Rs increased the Ca2+ transient of smooth muscle cells which may underlie the reduction of aorta contractions.

Conclusions

We conclude that the activation of TAS2Rs can reduce the tension of pre‐contracted rat aorta and the TAS2R agonists have the potential implication in the treatment of hypertension.

Support or Funding Information

Research Funding from Southwest University
Functional changes in vascular reactivity to adenosine receptors activation in type I diabetic mice.
Authors: Labazi Hicham , Teng Bunyen , Mustafa S. Jamal
Published: November 2023

Activation of adenosine receptors (ARs) have been implicated in a number of biological functions including cardiovascular and renal function. Diabetes is known to cause morphological and functional changes in the vasculature, resulting in abnormal responses to various stimuli such as adenosine. The aim for this study was to examine the effect of type I diabetes on the functional changes in conduit (aorta) and resistance arteries (mesenteric arteries, MA) to AR agonist's activation. Diabetes was induced in mice using streptozotocin (STZ, 10mg/Kg/day for 5 days; 12 weeks); concentration response curves (CRC) to acetylcholine, phenylephrine and selective agonists including A1AR (2‐Chloro‐N6‐cyclopentyladenosine, CCPA), A3AR (1‐[2‐chloro‐6‐ [[(3‐iodophenyl) methyl] amino]‐9H‐purin‐9‐yl]‐1‐deoxy‐N‐methyl‐β‐d‐ribofuranuronamide, Cl‐IBMECA), A2AAR (4‐[2‐[[6‐Amino‐9‐(N‐ethyl‐β‐D‐ribofuranuronamidosyl)‐9H‐purin‐2‐yl]amino]ethyl]benzenepropanoic acid hydrochloride, CGS‐21680) and A2BAR (2‐[[6‐Amino‐3,5‐dicyano‐4‐[4‐(cyclopropylmethoxy)phenyl]‐2‐pyridinyl]thio]‐acetamide, BAY 60‐6583) were performed in isolated aortae and MA from STZ and vehicle groups. Diabetes did not affect AR agonists‐mediated relaxation/contraction CRCs in MA. However, aortae from diabetic mice exhibited a significant decrease (p<0.05) in A1R‐mediated vasoconstriction. In addition, the aorta from STZ mice exhibited an increase in phenylephrine‐mediated contraction (EC50 6.88 ± 0.09 in STZ vs. 7.06 ± 0.09 in vehicle; p<0.05), while the relaxation to A2AAR agonist (CGS‐21680) tended to decrease in aortae from the STZ‐treated group, it was not statistically significant. Our data suggest that STZ‐induced diabetes caused functional changes in conduit arteries (decreased A1R‐mediated contraction), and these changes may be a compensatory to counterbalance the increased vascular contractility observed in diabetes.

Support or Funding Information

Supported by HL027339
Treatment with Captopril Enhances the Vascular Relaxation Induced by the Angiotensin II Type 2 Receptor (AT2R) Agonist CGP42112 in Diabetic Rats
Authors: Sabra Ramzi
Published: November 2023

The role of the AT2 receptor (AT2R) in physiological and pathophysiological processes is still controversial. Some studies suggest that expression of AT2R increases in certain diseases like diabetes mellitus. This could have beneficial effects as signaling through the AT2 receptor has been shown to counter the effects of angiotensin II mediated through the AT1 receptor, which are believed to contribute to pathological changes; this is supported by the favorable effects of ACE inhibitors and Angiotensin receptor blockers on the course of diabetes induced complications in renal function. We examined the effect of diabetes and of treatment with captopril on the vascular responses to CGP42112 (CGP), an AT2R selective agonist, in Sprague Dawley rats with streptozotocin (85 mg/kg i.v.) induced diabetes. Rats were followed for either 2, 4 or 12 weeks, at the end of which period thoracic aortas were isolated and used for measurement of relaxant responses to CGP42112 after pre‐contraction with phenylephrine. In a subset of the 4‐week study, osmotic pumps were implanted at week 2 to deliver either captopril (50 mg/kg/day) or losartan (5 mg/kg/day) s.c. for 2 weeks. All aortic ring studies were conducted in the presence of losartan (100 μM) to prevent interactions of CGP with the AT1R. CGP induced a mild (10%) relaxation in aortic rings at 2 weeks, 4 weeks and 12 weeks in the control animals. The response to CGP in aortic rings from diabetic rats was not different from controls. Treatment with losartan for 2 weeks did not alter the responses to CGP observed after 4 weeks. However, treatment with captopril for 2 weeks resulted in a markedly exaggerated relaxant response to CGP reaching up to 50% relaxation. These preliminary results suggest that while diabetes, per se, may not alter responses to CGP, treatment of diabetic rats (but not control rats) with captopril produces a marked increase in the sensitivity of the AT2R. Considering the known anti‐proliferative and anti‐ischemic effects of AT2R activation, these results potentially imply that the use of ACE inhibitors together with AT2R agonists could amplify the protective effect of ACE inhibitors against diabetes induced complications. Thus, the pathophysiological and therapeutic implications of this finding require further study.

Support or Funding Information

Supported by funds from the Medical Practice Plan, AUB Faculty of Medicine
Cardiac HMGB1/TLR4 Activation in Pre‐Type 2 Diabetes: A Potential Mechanism Triggering Diabetic Cardiovascular Complications
Authors: Vargas Elizabeth , Li Ying , Carrillo‐Sepulveda Maria Alicia
Published: November 2023

The prevalence of type 2 diabetes mellitus (T2DM) continues to increase at an alarming rate, to the point of epidemic standards, within the United States. One of the major, potentially lethal, complications of T2DM is the development of cardiovascular disease. Toll‐like receptor 4 (TLR4), a key component of innate immunity, has been implicated in diabetic complications due to its resultant increase in inflammation and oxidative stress. While previous findings from our lab have identified that vascular TLR4 plays a major role in DM vascular complications, the activation of TLR4 in the heart from pre‐T2DM has yet to be explored. We hypothesize that TLR4 signaling is activated in the heart of pre‐diabetic rats and is linked to an early inflammatory state. Male Goto‐Kakizaki (GK) rats, a non‐obese model of T2DM, were utilized in this study, with eight and sixteen week old animals representing the pre‐T2DM and T2DM groups, respectively. Wistar rats served as the non‐T2DM (control) group. As expected, blood glucose levels (mg/dL) were elevated in the T2DM group (162.75± 11.32 vs. 85.83 ± 8.25 control group, p<0.001, n=6). A slight increase in blood glucose levels were observed in the pre‐T2DM group (121 ± 7.75 vs. 85.83 ±8.25 control group, p<0.05). T2DM rats exhibited increased systolic blood pressure (SBP) (149± 12mmHg), an expected cardiovascular complication in T2DM, while pre‐T2DM rats displayed normal SBP (112 ± 7 mmHg). To determine whether pre‐T2DM conditions modulate TLR4 signaling in cardiac tissue, western blot analysis was performed. Cardiac TLR4 protein expression was markedly elevated in the pre‐T2DM group (3.1±0.1 fold increase vs. T2DM, p<0.01, n=4), paralleled by increased NFKβ expression (2.4 ± 0.2 fold increase vs. T2DM, p<0.05, n=4), an inflammatory marker and downstream target of TLR4 signaling. MyD88, an adaptor protein of TLR4 signaling, remained unaltered in cardiac tissue of pre‐T2DM subjects, suggesting that cardiac TLR4 activation in pre‐T2DM may occur via an MyD88‐independent mechanism. Of note, we found that high‐mobility group box 1 protein (HMGB1) expression was markedly increased in pre‐T2DM hearts (1.3 fold increase vs. control, p<0.01, n=4). HMGB1 is a damage‐associated molecular pattern (DAMP) molecule that has been suggested to be an endogenous TLR4 ligand. This finding implies HMGB1 may be a sterile ligand candidate in the activation of TLR4‐mediated inflammation in pre‐diabetic milieu. In conclusion, our results suggest that HMGB1/TLR4/NFKβ signaling pathway is activated in pre‐T2DM hearts. Therefore, strategies to target either HMGB1 or TLR4 pose a potential alternative therapy in the prevention of diabetic cardiovascular complications.

Support or Funding Information

NIH
SGLT1 Inhibition has Modest Gastrointestinal Consequences in Mice and Rats
Authors: Hornby Pamela J , Du Fuyong , Hinke Simon , Wallace Nathaniel , Cavanaugh Cassandre , Bender Corey , Jennis Matthew , Liang Yin , Gaul Michael , Kuo Gee Hong , Lenhard James
Published: November 2023

In the small intestine glucose is transported apically across the enterocyte by sodium/glucose cotransporter 1 (SGLT1; slc5a1) and its inhibition lowers the post‐prandial blood glucose excursion; however, questions remain about the effect of altered luminal glucose in the intestine. Using 13C6‐glucose LC/MS/MS detection we first quantified the inhibition of intestinal glucose uptake in SGLT1 −/− and −/+ mice in vivo, and pharmacologically by cmpd B (IC50 <20 nM for rSGLT1 & rSGLT2) in isolated rat jejunum mounted in Ussing chambers. After Cmpd B was dosed chronically, morphometric intestinal changes were assessed in Zucker Diabetic Fatty (ZDF) rats and cecal/fecal bacterial 16s RNA analysis performed in mice. A high fat diet In C57BL/6mice had variable effects on sglt1 mRNA expression (increased, decreased or not changed) in 3 separate experiments. Thus, there is no consistent impact of diet on SGLT1 mRNA expression. Jejunum mucosal SGLT1 mRNA expression was confirmed absent in −/− mice whereas +/− mice had ~ half the level of expression. After an oral 13C6‐glucose tolerance test (oGTT) the AUC was similar in WT and SGLT1 +/− mice, and reduced to 25% of AUC in −/− mice (−/−3,851±384, P<0.05 by ANOVA compared to −/+ 18,682±3,277 and WT 14,674±1,616; n=7–8/group). Apically administered 13C6‐glucose to rat isolated muscle‐stripped jejunum in the presence of cmpd B (1 μM) decreased the amount detected basolaterally (14.2 ± 1.8μM, n=16; versus 8.7 ± 1.3μM, n=12 after 90 mins). In ZDF rats treated daily with 0.3 or 1.0 mg/kg for up to 3 weeks, decreased HbA1c, and total GLP‐1 during an oGTT increased after the higher dose. Analysis of GI tissue showed no difference in jejunum mucosal SGLT1 or GLUT2 mRNA relative expression, and no gross mucosal adaption (villus height, villus width or mucosal weight). In C57BL/6mice, high sucrose diet or cmpd B in mice fed normal chow (1.0 mg/kg p.o. daily for 5 days) alone did not change cecal glucose concentration but, when combined with high sucrose diet, cecal glucose was increased (30.6 ± 3.4 mg/dL versus 17.1 ± 4.7 mg/dL, n=8/group). Switching to high sucrose diet changed cecal/fecal bacterial diversity by day 2, with some differences in bacterial relative abundance; however, neither microbial diversity nor abundance was correlated with cecal glucose concentrations. In conclusion, SGLT1 inhibition or genetic ablation reduces intestinal glucose uptake (although glucose is still absorbed). but any remaining excess luminal glucose is not associated with intestinal adaptation or bacterial population changes during the time frames studied.
Downregulation of Histone Deacetylase 9 (HDAC9) is Associated with Vascular Calcification in Diabetic Mice.
Authors: Benerofe Sara Arielle , Carrillo‐Sepulveda Maria Alicia
Published: November 2023

Vascular complications, including calcification, are some of the major problems faced by type 2 diabetic (T2DM) patients. In fact, the majority of these diabetic patients will die due to complications of vasculopathy. The current available anti‐diabetic treatment does not prevent the progression of vascular calcification; thus, new therapeutic approaches are urgently needed. Evidence has indicated that HDACs play a central role in the epigenetic mechanisms associated with diabetic complications. Recently, it was identified that a human polymorphism of HDAC9 is linked with acute coronary disease; however, it is still unknown whether HDAC9 is involved in vascular complications in diabetes. We hypothesize that diabetic conditions modulate HDAC9 expression, and this expression is associated with increased markers of vascular calcification. To address our hypothesis, we utilized aortas from fourteen‐week‐old male db/db mice, an experimental model of T2DM, and age‐matched heterozygous nondiabetic (ND) mice (db/m+). Aortas from db/db mice displayed significant increase in osteopontin (OPN) (2.0 fold increase, p<0.01, n=5) and bone morphogenic protein 4 (BMP4) (2.7 fold increase, p<0.05, n=5) compared with the control group, both classical markers of vascular calcification. Most importantly, these findings were correlated with significant downregulation of HDAC9 (1.1 fold reduction vs. control, p<0.05, n=4). Intriguingly, these results differ from previous reports that claim HDAC9 was increased in vascular complications, specifically atherosclerosis and coronary heart disease. Our results suggest that HDAC9 may be regulated differently in diabetic conditions and its downregulation may mediate the progression of vascular diabetic calcification. To further this investigation, we will be utilizing molecular approaches to overexpress HDAC9 in the vasculature to confirm the role of HDAC9 in the diabetic vascular complications.

Support or Funding Information

NIH
ANTITHROMBOTIC ACTIVITY OF NEW POTASSIUM SALT 2‐[3‐BROMINE‐1‐(THIETANIL‐3)‐1,2,4‐TRIAZOLIL‐5‐THIO]ACETIC ACID
Authors: Samorodov Aleksandr Vladimirovich , Kamilov Felix Khusainovich , Klen Elena Edmundovna , Khaliullin Ferkat Adelzyanovich , Khalimov Almaz Radikovich
Published: November 2023

The previous findings in vitro show high antiaggregation activity of newly synthesized potassium salt of 2‐[3‐bromine‐1‐(thietanil‐3)‐1,2,4‐triazolil‐5‐thio]acetic acid (substance I). The current findings are received in vivo on the model of collagen‐adrenaline thrombosis according to G.S. Di Minno subject to the FASEB Statement of Principles for the use of animals in research and education. Efficacy endpoint is the number of survived animals in comparison with the test group and comparator agents since the injection into the tail vein suspended matter of collagen and adrenalin upto the moment of death or during 14 days of monitoring. The survival analysis was carried out with the help of Kaplan‐Meier method. The difference in survival between the groups was assessed with the help of Wilkonson criterion generalized by Gehan. Intraperinoneal injection of the new secondary 1,2,4‐triazole reduced mortality in lab mice by 1,3 times (p<0,01) in comparison with the camparator agents (table 1). Thus, potassium salt of 2‐[3‐bromine‐1‐(thietanil‐3)‐1,2,4‐triazolil‐5‐thio]acetic acid is a potential antithrombotic agent.

The survival rate in the modeling of the collagen‐adrenaline thrombosis, M±SD

Subject
Control
Pentoxifylline
Aspirin
Substance I

Lifetime, h
2,0±1,8
165,8±157,6*
172,6±102,8*
206,9±135,2*p2<0,001p3<0,005

p1<0,01 ‐ control vs experimental group;

p2 ‐ substance I vs pentoxifylline, p3 ‐ substance I vs aspirin, χ2=33,1.
Polyphenol Rich Extract of Syzygium cumini (L.) Skeels Leaves Decreases Platelet Aggregation and Inhibits Reductase Activity of Protein Disulfide Isomerase.
Authors: da Silva Samira Abdalla , Sousa Hiran Reis , Gaspar Renato , Fontelles João Lucas , Trostchansky Andrés Ezequiel , Paes Antonio Marcus
Published: November 2023

Platelet agreggation results from conformational changes in αIIbβIII integrin, which are dependent on protein disulfide isomerase (PDI) activity. Recently, PDI inhibition has been proposed as an antithrombotic mechanism, for that polyphenolic compounds have emerged as potential inhibitors. Our group has demonstrated that Syzygium cumini (L.) Skeels (Myrtacea) leaf contains multiple polyphenols, such as: gallic acid, miricetyn‐3‐a‐arabinoside, mirycetin deoxiexoside and quercetin‐3‐glycoside; which enable this species for anti‐platelet activity. Therefore, this study aimed to evaluate the effects of polyphenol rich extract (ERP) from S. cumini leaf on platelet aggregation and PDI reductase activity. Platelet‐rich plasma (PRP) from healthy volunteers (n=5) was incubated with ERP for 25 min at concentrations ranging from 10 to 1000μg/mL, previously to activation with 2.5 or 5μM ADP. ERP at the higher concentration inhibited platelet aggregation in 66 and 33%, respectively. Under thrombin stimulus (0.01 and 0.02 U/mL), ERP (1000 μg/mL) reduced platelet aggregation in ca. 35%. Moreover, ERP also inhibited PMA‐induced platelet aggregation by 29% in its higher concentration. To analyze the ERP effect on αIIbβIII integrin activation, flow citometry protocols were conducted in washed platelets similarly pre‐treated with ERP (10, 100 and 1000μg/mL) before activation with thrombin (0.02U/mL) and tagged with PAC–1 FITC antibody. ERP dose‐dependently reduced the integrin activation in presence of 10 μg/mL (21%), 100 μg/mL (27%) and 1000 μg/mL (33%). Finally, ERP effects on PDI reductase activity were also assessed at concentrations ranging from 0.1 to 100 μg/mL. A maximum inhibitory effect on PDI reductase activity of 77% was found for the higher concentration. Therefore, our data supports that ERP‐containing polyphenols from S. cumini leaf reduce platelet aggregation, probably through PDI inhibition, which demonstrates its prominent anti‐platelet property.

Support or Funding Information

Financial support: FAPEMA; UFMA
Terminalia methanol extracts inhibit the amidolytic activity of human activated factor XII (Hageman factor)
Authors: Madkhali Hassan , Alharbi Abdullah , Ali Zulfiqar , Khan Ikhlas , Shariat‐Madar Zia
Published: November 2023

The long‐term goal of this study is to develop the selective inhibitors of FXIIa based on traditional medicine, which might be safe, and effective. There are positive correlations between increased plasma FXIIa levels with coronary heart disease as well as increased risk of recurrent coronary events. Although they maintain a normal hemostasis, FXII knockout mice (FXII−/−) display defective arterial thrombus formation. In addition, FXII−/− mice are protected from development of cerebral ischemia and pulmonary embolism. Thus, inhibition of FXIIa would help prevent cardiovascular diseases. The present study examines the effects of different but related herbal medicines that impact on the plasma blood coagulation FXII. While it initiates the activation of blood coagulation to stop any bleeding, FXII helps the remaining blood clotting factors and platelets to keep certain circulating pathogenic microorganism close to the phagocytes. The negative charge on the microorganism surface or platelet polyphosphate (poly P) binds readily to zymogen FXII, resulting in the activation of FXII. Evidence indicates that poly P containing pathogens promote FXII‐mediated blood coagulation independent of factor XI, reflecting a novel poly P‐induced thrombus formation. The methanol extracts of T. chebula fruit, T. bellirica fruit, T. arjuna fruit, and T. arjuna bark blocked FXIIa activity with IC50 values of 30, 50, 190, 190 and 27 mg/ml, respectively. Two isolated compounds from Terminalia, Arjunoglucoside II and arjunic acid blocked FXIIa activity with IC50 values of 370 and 780 μM, respectively. T. chebula fruit, T. and arjuna fruit extracts at 50 mg/ml did not affect endothelial cells viability. Overall, T. chebula fruit and T. arjuna fruit extracts might be useful to improve symptoms of FXIIa‐dependent inflammation and thrombosis.
Histone Modification is a Novel Epigenetic Mechanism to Up‐Regulate Human Blood‐Brain Barrier Transporters
Authors: You Dahea , Wen Xia , Morris Ayeshia , Richardson Jason R , Aleksunes Lauren M
Published: November 2023

Multidrug Resistance Protein 1 (MDR1, ABCB1) and the Breast Cancer Resistance Protein (BCRP, ABCG2) expressed at the blood‐brain barrier (BBB) are key efflux transporters that extrude chemicals from the BBB and regulate the efficacy and/or toxicity of chemicals in the brain. Prior studies in cancer cells have pointed to the ability of histone deacetylase (HDAC) inhibitors to modulate the expression and function of MDR1 and BCRP. However, whether or not such regulation occurs at the BBB is not known. Here we sought to test whether HDAC inhibitors could potentially alter expression and function of MDR1 and BCRP at the BBB. To test this, we treated immortalized human brain capillary endothelial (hCMEC/D3) cells, a model of the BBB, with six different HDAC inhibitors, valproic acid (VPA), sodium butyrate (NaB), romidepsin, apicidin, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA), and assessed for expression and function of MDR1 and BCRP. HDAC inhibition following treatment was confirmed by increased levels of acetylated histone H3 protein. After 12 h of treatment, VPA, apicidin, SAHA, and TSA up‐regulated MDR1 mRNA levels between 50% and 200%. All six HDAC inhibitors significantly induced BCRP mRNA levels between 100% and 270%. Similarly, the protein expression of MDR1 and BCRP transporters was up‐regulated about two‐fold at 24 h. Enhanced MDR1 expression corresponded with reduced intracellular accumulation of the substrate rhodamine 123. Collectively, these results demonstrate that HDAC inhibitors up‐regulate MDR1 and BCRP transporters at the BBB by modifying histone acetylation. The clinical use of HDAC inhibitors may enhance efflux transporter activity at the BBB and restrict access of xenobiotics to the brain.

Support or Funding Information

Supported by R01ES021800, P30ES005022.
Novel Characterization of Docohexanoic Acid and Its 12‐LOX Products, 11‐HDHE and 14‐ HDHE on Human Blood Platelets
Authors: Hawley Megan , Craven Da'Quan Dominique , Edokobi Nnamdi , Holman Ted , Holinstat Michael
Published: November 2023

Cardiovascular disease (CVD) is currently the leading causes of mortality in the United States in both men and women. Thrombotic‐associated ischemic injuries due to excessive platelet activation are largely responsible for the high level of death associated with CVD. Therapies that seek to pharmacologically inhibit platelet activation, aggregation, or clot stabilization, have the potential to reduce the high mortality rates associated with CVD. Fish oils are currently taken as a cardioprotective supplement, and consist of a mixture of two n‐3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Data elucidating the mechanism by which PUFAs interact with platelets remains controversial. The oxygenated products of PUFAs—oxylipins—have been shown to be critical regulators of platelet function. Our lab recently illustrated the essential role of 12‐lipoxygenase (12‐LOX) in the ability of another PUFA, dihomo‐γ‐linolenic acid (DGLA), to inhibit platelet activation and thrombosis via its oxylipin, 12(S)‐hydroxy‐8Z,10E,14Z‐eicosatrienoic acid (12(S)‐HETrE). Previously, it has been shown that DHA has antiplatelet activity; therefore, we hypothesize that 12‐LOX oxylipins are responsible for the antiplatelet effects of DHA. Our study sought to investigate the role that 12‐LOX derived products of DHA, 11‐ and 14‐ hydroxydocosahexaenoic acid (11‐ and 14‐ HDHE), have on human blood platelet activation. Platelet aggregation, calcium release, granule release, and integrin aIIbb3 activation were measured following agonist stimulation in the presence of 11‐ or 14‐ HDHE. Our data demonstrate that 11‐ or 14‐ HDHE dose‐dependently inhibited aggregation in response to submaximal concentrations of either thrombin or collagen. Additionally, we confirmed that 11‐ or 14‐ HDHE impinged on intracellular signaling as demonstrated by a decrease in Ca2+ mobilization, as well as granule secretion in platelets treated with 11‐ or 14‐ HDHE compared to DMSO treated platelets. Ongoing studies will help to elucidate the mechanism by which DHA and its metabolites inhibit platelet function. Once completed this will be the first study to clearly define a role for DHA and its 12‐LOX metabolites in the regulation of platelet aggregation.
11/14‐oxy‐DPA, the 12‐LOX derived metabolites of DPA, inhibit platelet activation through the cGMP‐PKG signaling pathway
Authors: Yeung Jennifer , Edokobi Nnamdi , Hawley Megan , Craven DaQuan , Li Cecilia , Holman Theodore R. , Holinstat Michael
Published: November 2023

Platelet‐mediated thrombosis is the primary underlying mechanism leading to cardiovascular life‐threatening clinical events. Regulating excessive platelet reactivity is an essential aspect of antithrombotic therapy. Previously, we had shown that lipid metabolites of 12‐lipoxygenase (12‐LOX) such as 12‐HETrE, a 12‐LOX oxylipin of the ω‐6 polyunsaturated fatty acid dihomo‐gamma‐linolenic acid, could function as an inhibitor to platelet activation. Here, we demonstrate for the first time that another ω‐6 PUFA, docosapentaenoic acid (DPA; 22:5n‐6), can be endogenously oxidized by 12‐LOX to generate two distinct oxylipins, 11‐oxy‐DPA and 14‐oxy‐DPA to regulate platelet reactivity. Although both 11‐oxy‐DPA and 14‐oxy‐DPA were able to significantly inhibit platelet activation, we observed differences in the potency of the 12‐LOX‐derived metabolites of DPA,n‐6. 11‐oxy‐DPA was able to significantly inhibit platelet aggregation at a lower concentration than 14‐oxy‐DPA. We also observed calcium mobilization was significantly attenuated in the presence of 11‐oxy‐DPA or 14‐oxy‐DPA. Furthermore, to determine whether 11‐oxy‐DPA and 14‐oxy‐DPA could be regulating platelet function through a Gαs signaling pathway by which it could be behaving similarly to prostacyclin or 12‐HETrE, we measured a downstream effector of cAMP, the phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at serine 157 by PKA. We observed no differences in VASP phosphorylation at serine 157 in platelets following 11‐oxy‐DPA or 14‐oxy‐DPA treatment compared to vehicle control. However, VASP phosphorylation at serine 239, a substrate of PKG, was enhanced in platelets treated with 11‐oxy‐DPA or 14‐oxy‐DPA treatment. Thus, this suggest that 11‐oxy‐DPA and 14‐oxy‐DPA inhibit platelet activation through a novel cGMP‐PKG‐dependent pathway. This is the first study to demonstrate the novel metabolites of ω‐6 DPA generated by 12‐LOX have role in regulating platelet activation through the cGMP‐PKG signaling pathway and expands potential targets for future therapeutic intervention for the prevention of thrombotic events leading to myocardial infarction and stroke.

Support or Funding Information

F31 HL129481

GM105671
Switching off TRPC6 Signaling: A new anti‐edemagenic strategy
Authors: Amin Md Ruhul , Piplani Honit , Sharma Tiffany , Mehta Dolly
Published: November 2023

Transient receptor canonical channel (TRPC)‐6, ubiquitously expressed nonselective cation channel activated by second messenger DAG, induces calcium entry in endothelial cells independent of ER store depletion. Recently, we have shown that deletion of TRPC6 in mice prevented lung endothelial permeability and inflammation in response to thrombin and LPS indicating targeting TRPC6 will be valuable in preventing uncontrolled increase in endothelial permeability. TRPC6 activity has been implicated in acute lung injury sepsis, pulmonary hypertension and focal segmental glomerulosclerosis (FSGS). Here we identified key motif in Ankyrin 1 domain of TRPC6 that regulates gating of the channel. Amino acid sequence analyses of TRPC6 revealed the presence of 4 ankyrin‐like repeat domains which are known to be important for protein‐protein interactions, channel assembly and activity. Thus, we performed mutational analysis on these repeats and assess channel activity. We found that deletion of Ist ankyrin repeat in TRPC6 blocked calcium influx in HEK cells. Further mutational analysis identified that 96–111 residues within Ist ankyrin repeat was required for TRPC6 cell surface retention and calcium influx. Sequence analysis using ELM database predicted YGNI (aa108‐aa111) motif as the one likely regulating TRPC6 activity based on resemblance with adaptor protein complex, mu subunit (AP2M1), an important signal for endocytosis. Mutation of YGNI to AAAA blocked Ca2+‐entry in response to Hyperforin and OAG. Based on above studies, we have developed specific peptides which efficiently blocked Ca2+‐entry in response to Hyp9 in both HEK and HPAE cells. Ongoing studies are underway to establish the clinical relevance of TRPC6 activity blocking peptides in animal model of lung injury.
Tetramethylpyrazine reduces intra‐platelet calcium via suppressing the SGK1/STIM1/Orai1 pathway
Authors: Wang Zhentao , Cang Hongbo , Gao Shuibo , Han Lihua , Wu Hong
Published: November 2023

Tetramethylpyrazine (TMPZ) is frequently administered for cardiovascular diseases. Although TMPZ is thought to inhibit the elevation of intra‐platelet calcium ions (Ca2+), the underlying mechanism remains unknown. Herein, the time‐dependent effect of TMPZ on adenosine diphosphate (ADP)‐induced platelet aggregation was assessed using a turbidimetric approach. Meanwhile, the intracellular Ca2+ concentration following ADP stimulation was determined using a fluo‐4 Ca2+ probe. We then clarified the mechanisms underlying TMPZ intervention by determining the mRNA and protein levels of Ca2+‐related pathway including stromal interaction molecule l (STIM1), Orai1, and serum/glucocorticoid‐regulated protein kinase 1 (SGK1) by real‐time PCR and Western blot, respectively. The results showed that TMPZ significantly reduced platelet aggregation in a dose‐dependent manner. In addition, TMPZ markedly decreased intracellular Ca2+ concentration following ADP stimulation. Treatment with 2‐mM TMPZ inhibited ADP‐induced expression of STIM1 and Orai1 in platelets at both the mRNA and protein levels, and also reduced the expression of the SGK1 protein. In summary, TMPZ reduces the intra‐platelet Ca2+, which then inhibits platelet activation and thrombosis. The underlying mechanism is presumed to be through the suppression of the SGK1/STIM1/Orai1 pathway.

Support or Funding Information

This work was supported by grants from the Scientific Research Foundation for the Returned Oversea Chinese Scholars, State Ministry of Education of China and Collaborative Innovation Center of Henan University of Traditional Chinese Medicine
Circulating Mitochondrial (mt) DNA Fragments as Precursors of Inflammation in Early Stages of Atherogenesis
Authors: Ruchko Mykhaylo V. , Pastukh Viktor M. , Sukhanov Sergiy , Gorodnya Olena M. , Danchuk Svitlana D. , Gillespie Mark N.
Published: November 2023

Atherosclerosis and its vascular complications are the leading cause of morbidity and mortality among Western populations. It is widely accepted that inflammation plays a major role in all stages of atherogenesis, including its initial phases. However, the mechanisms of initiation of inflammatory response predisposing to the development of atherosclerosis are not well understood. Mitochondrial dysfunction underlies many key processes in the development of atherosclerosis, including generation of reactive oxygen species, apoptosis, and inflammation. There is growing evidence that oxidative mtDNA damage can directly promote atherosclerosis. The mechanism linking mitochondrial damage to atherogenesis remains unclear, but may include initiation of inflammation by circulating mtDNA fragments known as DAMPs (Damage Associated Molecular Patterns). Previously, we have demonstrated that mice deficient in DNA repair glycosylase Ogg1 and fed a high fat diet developed larger fatty streaks in the aortic valve as a sign of early atherogenesis that was correlated with higher mtDNA oxidative damage in cardiac tissue and elevated content of plasma mtDNA fragments in comparison to wild type animals. In the present study we tested the hypothesis that development of atherosclerosis in animal model is associated with increased abundance of circulating pro‐inflammatory mtDNA fragments. ApoE‐null and wild type mice of increasing age were analyzed for atherosclerotic burden within the aortic valve and for abundance of mtDNA fragments in plasma. ApoE‐null mice develop atherosclerotic plaque in the aortic valve either spontaneously with age or after feeding a high fat diet. Early lesions were detected in ApoE‐null mice by 3 months of age whereas in 5‐month old animals they became prominent. Atherosclerotic burden reached 30% of valve area in 10‐month old animals and remained at that level in 12‐month old mice. Wild type animals did not develop lesions within the aortic valve at any age. The abundance of circulating mtDNA fragments was higher in ApoE‐null mice in all age groups when compared to wild type animals. Importantly, the largest relative increase in mtDNA fragments (5 to 6.5 fold vs. age‐matched controls) was observed in ApoE‐null mice at 3 and 5 months. These results indicate that circulating mtDNA fragments could be precursors of inflammation development in early stages of atherogenesis and suggest that efficient maintenance of mtDNA integrity could be an important target for atheroprotection.

Support or Funding Information

NIH R01 HL113614
Functional Analyses of Reticulated Platelets
Authors: Hayman Melissa , Armstrong Paul , Chan Melissa , Warner Timothy
Published: November 2023

Objective

To isolate reticulated platelets from platelet rich plasma and consequently assess functional reactivity and mRNA content.

Introduction

The platelet population is a heterogeneous mix of various sub‐populations, potentially each with distinct biological capabilities. Of particular interest is the newly formed reticulated sub‐population of platelets, so called because of the presence of RNA in their cytoplasm. This subpopulation is estimated to account for roughly 10% of the total platelet population and has been reported to have a higher thrombotic potential than the non‐reticulated population.

Methods

Flow cytometry was used to identify reticulated platelets within platelet rich plasma following staining with thiazole orange. The top 10% of thiazole orange positive cells was selected as the ‘reticulated population’, the middle 50% as the ‘intermediate population’ and the bottom 30% as the ‘non‐reticulated population’. mRNA content in each of the subpopulations was determined using qRT‐PCR. Platelet aggregation and P‐selectin expression, in response to various agonists, were also assessed using flow cytometry. Imaging flow cytometry permitted the localisation and relative assessment of the contribution of both reticulated and non‐reticulated platelets to the formation of aggregates.

Results

Reticulated platelets were confirmed to contain more mRNA than either the intermediate or non‐reticulated populations (average Ct value: PF4, 23.9 vs 27.9 vs 29.9; TUBB1, 25.7 vs 30.4 vs 32.8; ITGA2B, 27.0 vs 31.9 vs 33.9). Following exposure to platelet agonists the percentages of reticulated platelets within the single platelet population were significantly reduced compared to vehicle (AA 1 mM, 2.4% vs 9.4%; ADP 20 μM, 6.2% vs 9.6%; collagen 1 μg/mL, 6.0% vs 9.1%; TRAP‐6 25 μM, 1.3% vs 9.1%). Image analysis also indicated that reticulated platelets were more often present at the centre of platelet aggregates. Of all formed aggregates, reticulated platelets were present in 95±2% and non‐reticulated platelets in 42±6%. In addition, reticulated platelets had a significantly higher fold change in the expression of cell surface P‐selectin (CD62P) than non‐reticulated platelets following exposure to TRAP‐6 (2.5 μM, 2.6±0.4 fold greater; 25 μM, 2.7±0.4 fold greater; p=<0.05).

Conclusion

Upon activation reticulated platelets, i.e. those containing more mRNA, are disproportionally recruited to the centre of forming aggregates demonstrating that this platelet sub‐population are important to the early stages of aggregate formation. Thus reticulated platelets present an interesting target for research as they are more reactive than non‐reticulated platelets and may be early drivers of thrombus formation.

Support or Funding Information

Work was generously supported by Barts and the London School of Medicine and Dentistry and The Wellcome Trust (101604/Z/13/Z).
The Role of Leptin in Coronary Collateral Growth
Authors: Graham Kathleen , Ohanyan Vahagn , Enrick Molly , Kolz Christopher , Chilian William M. , Yin Liya
Published: November 2023

Leptin is an adipokine that is conventionally thought to have its major effects on appetite suppression, but more recently it is suggested to have a variety cardiovascular effects including on vascular growth. The Zucker obese fatty (ZOF) rat is a model of dysfunctional leptin signaling due to a mis‐sense mutation of the leptin receptor. This mutation leads to hyperphagia inducing dietary‐induced obesity and phenotypes consistent with the metabolic syndrome, e.g., obesity, hypercholesterolemia, hyperglycemia, and hyperlipidemia. Previously we observed abrogated coronary collateral growth (CCG) in ZOF rats, but we did not determine if this deficiency is directly due to the mis‐sense mutation in leptin, which could negatively impact the vasculature, or indirectly related to the hyperphagia‐induced metabolic syndrome. To resolve between these two possibilities, we studied CCG in young ZOF rats prior to the development of obesity and the alterations in blood chemistries. CCG was stimulated by repetitive episodes of myocardial ischemia (RI) induced by chronic implantation of a pneumatic snare around the left anterior descending artery (LAD), which was performed during a left thoracotomy. Pressurization of the snare induced occlusion of the LAD and a total of 24 occlusions were performed throughout the day. CCG in rats was assessed by the measurement of myocardial blood flow (MBF) using contrast echocardiography at Day 0 (prior to the initiation of RI) and after 10 days of RI in the normal and collateral‐dependent regions; and is presented as a ratio of collateral zone/normal zone flow (CZ/NZ). In old (6–9 months, n=8) and young (3–4 months, n=4) ZOF rats, the CZ/NZ ratios at Day 0 were 0.15±.03, and 0.22±0.04, respectively (P=NS). This indicates that the native collateral circulation was comparable in the two groups. In contrast, following 10 days of RI, the CZ/NZ flow ratio was unchanged (from Day 0) in the old ZOF rats (0.17±0.05), but significantly increased in the young ZOF rats (0.78±0.07; P<0.05 vs Day 0 and old ZOF). To eliminate the possibility that the differences are related to the ages of the ZOF rats, age‐matched Lean controls were studied. The CZ/NZ ratios were equal in the young and old lean rats suggesting that the difference in the ZOF rats is not related to age. Based on these results we conclude that the reason why CCG is impaired in ZOF rats is not due to an absence of leptin signaling; rather the impaired coronary collateral growth in ZOF rats is likely related to the myriad cardiovascular risk factors associated with the metabolic syndrome.

Support or Funding Information

This work was supported by NIH/NHLBI (HL100828Z and HL83366), Fibus Family Foundation, Frances Schermer Charitable Trust & The Lillian Schermer Charitable Trust, and the Joy Cone Company to W.M.C.; NIH/NHLBI (HL115114) to LY.
Neutrophil Extracellular Traps Contribute To Endothelial Barrier Dysfunction During Sepsis
Authors: Meegan Jamie E , Beard Richard S , Yang Xiaoyuan , Reynolds Jason J , Coleman Danielle C , Jannaway Melanie , Yuan Sarah Y
Published: November 2023

Neutrophil extracellular traps (NETs) are DNA‐webs containing histones and granular enzymes produced by neutrophils during host defense against infection. It is becoming increasingly apparent that circulating NETs exacerbate tissue injury associated with inflammation. The purpose of this study was to determine the effects of NETs on endothelial barrier function. We identified increased markers of NETs in the lung and plasma of mice subjected to polymicrobial septic injury induced by cecal ligation and puncture. In an intravital microscopic analysis of microcirculation, NETs were found in mesenteric microvessels in response to fMLP stimulation. In cultured endothelial cell monolayers, treatment with NETs produced by activated human neutrophils induced barrier dysfunction as indicated by reduced transendothelial electrical resistance and increased albumin transendothelial flux. Exogenous histones produced a similar effect on barrier dysfunction. Immunofluorescence confocal microscopy indicates the involvement of conformational changes in adherens junction proteins VE‐cadherin and β‐catenin as a potential mechanism underlying NET‐induced barrier dysfunction. Furthermore, myosin light chain phosphorylation in endothelial cells was observed after NET stimulation, supporting the involvement of cytoskeleton response in this process. Taken together, the data suggest NETs may contribute to endothelial barrier dysfunction during inflammatory response associated with neutrophil activation.

Support or Funding Information

This work is supported by NIH RO1s HL126646, HL070752, and GM097270.
Super resolution imaging visualized the nanoscaled localization of integrin activation during human neutrophil arrest
Authors: Fan Zhichao , Kiosses William Bill , Ley Klaus
Published: November 2023

Adhesion on vascular endothelium is the vital step for leukocyte recruitment and inflammation. Neutrophil is the most abundant leukocyte and is essential for innate immunity and inflammation. The activation of beta2 integrin is critical for neutrophil adhesion. Integrins are bidirectional signaling molecules that are bent at rest. Upon cell activation, integrins can extend (E+) and acquire a high affinity conformation with an “open” headpiece (H+). The canonical “switchblade” model of integrin activation proposes that the E+ conformation precedes H+, and the two are thought to be structurally linked. Only the E+H+ conformation can mediate cell adhesion by binding to ligand in trans. In our previous study, we found out an unexpected bent‐open (E−H+) conformation and an alternative allosteric pathway, which integrin can H+ prior to E+, during human neutrophil arrest. However, the resolution of our previous quantitative dynamic footprinting (qDF) microscopy is limited by optical diffraction limit (~300nm) that we cannot accurately observe the conformation of signal integrin, which is under nanoscale (~20nm). By combining stochastic optical reconstruction microscopy (STORM) with our qDF microscopy, we acquired nanoscale super‐resolution images of integrin activation on arrest human neutrophils. These images confirmed our finding that E−H+ integrin exist and brought us the accurate number and precise local distribution of E−H+, E+H− and E+H+ integrins on arrest neutrophils. We found out that E+H+ integrins are mostly located in microvilli tips and form microscale clusters to support neutrophil arrest.

Support or Funding Information

This research was supported by funding from the National Institutes of Health, USA (NIH, HL078784) and WSA postdoctoral fellowship from the American Heart Association, USA (AHA, 16POST31160014).