bluuebunny/crossref_metadata_2025 huggingface dataset:
Refining the Half‐Site Reaction Mechanism of SULT1B1 Using Engineered Heterodimers
Authors: Tibbs Zachary Evan , Falany Charles N
Published: November 2023

The cytosolic sulfotransferases (SULTs) biotransform drugs and hormones into sulfate esters, aiding in the deactivation and/or excretion of the compounds from the cell and body. Each human SULT isoform is a homodimer interfacing along a conserved motif with the consensus sequence KxxxTVxxxE. Despite conservation of the dimerization domain across all fourteen human SULT isoforms, its contribution to SULT activity is not fully understood. Based on results from recent molecular dynamic simulations (MDS) of dimeric SULT1B1 in the presence and absence of the cofactor, 3′‐phosphoadenosine 5′phosphosulfate (PAPS), our laboratory has developed a mechanistic model for SULT dimer half‐site reactivity. In this proposed reaction mechanism, the binding of PAP(S) to the enzyme surface elicits structural changes which are communicated to the partnering subunit through the dimerization domain. The result of this communication is a dimer with a single active subunit at any given time. The dimer subunits oscillate between active and inactive states, “taking turns” as the active subunit. The advantage of this oscillating mechanism is thought to be an overall increased reaction rate, primarily propelled by a more favorable koff for the reaction byproduct, 3′ 5′‐diphosphoadenosine (PAP). As a means for testing the oscillating activity hypothesis in vitro, we sought to disrupt the activity of one subunit of the dimer while leaving the activity of the neighboring subunit fully in tact. Comparison of the heterodimer's kinetic parameters to those of the wildtype isoform would discern the involvement of the partnering subunit in the overall reaction mechanism. Two key amino acid residues were mutated to disrupt the activity of SULT1B1. Mutation of Arg258 to a Lys rendered the SULT deficient in its ability to bind PAPS (Kd ≈ 200 uM), whereas mutation of the catalytic base, His109, to a Tyr rendered the SULT incapable of catalyzing sulfonate transfer while leaving substrate binding unaffected. Unfortunately, the favorable kon for SULT dimerization complicated isolation of heterodimeric SULT species (only one subunit with the desired mutation). To overcome this limitation, we engineered SULT1B1 isoforms capable of selective dimerization, propelled by amino acid charge repulsion/attraction properties. This heterodimer‐generating system allowed us to manipulate each individual subunit independently prior to dimerization. Therefore, we combined the heterodimer‐generating system with the activity‐altering mutations (R258K and H109Y) to isolate SULT dimers with one fully active subunit. The resulting heterodimeric SULT isoforms are currently the subjects of in vitro sulfation activity analyses.

Support or Funding Information

NIH Grants: GM113980 and ES022606
Effects of Cimaglermin alfa Administration on LV Function in Rats with Chronic Heart Failure Induced by LAD Occlusion
Authors: Ganguly Anindita , Zolty Ronald , Troy Erika , Iaci Jennifer , Hackett Craig , Caggiano Anthony , Parry Tom J
Published: November 2023

Introduction

Neuregulin growth factors are essential for the normal development and function of the heart. Neuregulins have been found to protect and restore left ventricular (LV) function in a variety of species with heart failure (HF) induced by myocardial infarction (MI). No report so far has shown the effect of neuregulin administration on LV function when treatment is delayed until 20‐weeks post‐MI in rats.

Aim

The goal of this study was to examine the effects of delayed treatment with the neuregulin, cimaglermin, on LV function (echocardiographic) and characterize the effects of withdrawal and re‐initiation of treatment in rats with chronic HF induced by LAD ligation.

Methods

LAD ligation was performed in (n=10–12/group) 8–10 week old male Sprague Dawley rats. At 20 weeks post‐ligation, rats were randomly assigned to vehicle or cimaglermin (0.5 and 2.6 mg/kg IV or 2.6 and 10 mg/kg SC) treatment twice weekly for 1 month. This was followed by a 1 month treatment‐free period and a re‐treatment period similar to the first. Echocardiographic parameters were assessed at least once weekly throughout the study.

Results

Ejection fraction (%EF) and end systolic volume (ESV) improved with intravenous and subcutaneous cimaglermin treatment. Both %EF and ESV reverted to vehicle‐treated levels during a 4 week washout of cimaglermin with both routes of administration. At an intravenous dose of 0.5 mg/kg, the changes in %EF and ESV were significantly different from vehicle control during the re‐treatment period. At 2.6 mg/kg IV, the %EF (see graph) and ESV improved significantly during both treatment periods (p<0.05). For subcutaneous dosing, %EF improved significantly vs vehicle treatment during the initial and re‐treatment periods (p<0.05) at both SC doses while ESV trended toward improvement compared to vehicle controls at the high SC dose.

Conclusions

Cimaglermin treatment initiated at 20 weeks post‐MI produced significant improvement in LV function in rats. Cimaglermin re‐treatment following a 4 week withdrawal period produced LV functional improvements similar to those seen with initial treatment.
Pharmacological Perturbations of Eicosanoids in vitro and ex vivo in a Novel, Broad‐spectrum, Plasma Lipidomics Assay
Authors: Mazaleuskaya Liudmila L , Ricciotti Emanuela , Lawson John A , Li Xuanwen , Grant Gregory , Grosser Tilo , FitzGerald Garret A
Published: November 2023

Eicosanoids are a family of bioactive lipids formed from arachidonic acid and include prostaglandins, leukotrienes and related compounds. Nonsteroidal anti‐inflammatory drugs (NSAIDs) suppress prostaglandins and are widely used for anti‐pyretic, analgesic and anti‐inflammatory effects. NSAIDs selective for inhibition of cyclooxygenase (COX)‐2 have fewer gastro‐intestinal side effects, but predispose patients to increased cardiovascular risk. The cardiovascular complications prompted interest in microsomal prostaglandin synthase 1 (mPGES‐1), downstream of COX‐2, as a novel drug target. Unlike COX‐2, global deletion of mPGES‐1 in mice suppresses prostaglandin E2 (PGE2) and augments prostacyclin (PGI2) through substrate rediversion, and does not predispose to thrombogenesis and hypertension. However, cell‐specific deletions of mPGES‐1 reveal that the predominant prostaglandin product of substrate rediversion varies by cell type. We hypothesized that inhibition of mPGES‐1 leads to changes in bioactive lipids beyond the prostaglandin pathway and aimed 1) to develop a mass spectrometry‐based, broad‐spectrum lipidomics approach to measure arachidonate‐derived products additional to prostanoids and 2) to develop a human whole blood assay (hWBA) to study substrate rediversion upon mPGES‐1 vs COX‐2 inhibition. We have developed a novel, reproducible, analytical method to scan the impact of drugs on the bioactive lipidome in human whole blood in vitro and ex vivo. We observed substrate rediversion in both COX and LOX (lipoxygenase) pathways after mPGES‐1 inhibition, which was distinct from inhibiting COX‐1/2. We validated our in vitro assay by its application ex vivo after administration of a COX‐2 inhibitor to human subjects. Overall, we have shown that blockade, alone and in combination, of diverse targets in the eicosanoid synthesis/response cascade results in both expected and unanticipated signals in the circulating human lipidome. Such novel aspects of drug action prompt reconsideration of how drugs in these pathways might be utilized in the alleviation of disease.

Support or Funding Information

This study was supported by The Personalized NSAID Therapeutics Consortium (PENTACON: HL117798) and by T32 H107971.
Angiotensin II Induces Prostaglandin E2 Production and Oxidative Stress in the Renal Cortex
Authors: Alanazi Wael Abdullah , Fakhruddin Selim , Jackson Keith
Published: November 2023

Angiotensin II (AngII) is a systemic vasoconstrictor and body fluid regulator. Virtually all released AngII binds to AT1 receptors, which stimulates cell signaling pathways in various cell types. In kidneys, AngII enhances arachidonic acid release by increasing the activity of phospholipases enzymes and cyclooxygenase‐2 (COX‐2), leading to an elevation of PGs predominantly prostaglandin E2 (PGE2). Thus, the current study hypothesizes that acute AngII infusion induces the local production of PGE2 and oxidative stress in the renal cortex, which results in renal damage. Male Sprague Dawley rats (200 – 300g) were anesthetized and microdialysis probes were placed in the renal cortex. After a stabilization period, a 4 hour control period was followed by vehicle or AngII (7ng/kg/min, IV) treatment for 4 hours. Blood pressure was elevated significantly during AngII infusion from (109.8 ± 0.085mmHg) to (129.36 ± 0.1mmHg). Urine flow, urinary Na+/K+ excretion, glomerular filtration rate, and renal blood flow were decreased in comparison with control. Through renal microdialysis, we found that PGE2 levels were significantly induced by AngII treatment when compared to control. In addition, acute AngII infusion augmented the present oxidative stress in the renal tissues. Superoxide and peroxynitrite levels were evaluated by EPR using CPH and CMH as spin traps. In the current study, we found that local effects of acute AngII infusion stimulate PGE2 and free radical release producing a disruption in normal renal hemodynamic function. In summary, the current study demonstrates a potential mechanism by which acute elevations in AngII promotes hypertension induced end organ kidney damage.

Support or Funding Information

Saudi Arabian Cultural Mission (SACM), INBRE
Focally Acting Thrombin‐Inhibiting Nanoparticles Forestall Atherosclerosis in ApoE‐null Mice
Authors: Palekar Rohun U , Jallouk Andrew P , Myerson Jacob W , Pan Hua , Wickline Samuel A
Published: November 2023

Background

Thrombin plays a central role in mediating both thrombosis and inflammation through cleavage of its receptor, protease activated receptor 1 or PAR‐1. Activation of PAR‐1 results in the expression of numerous procoagulant and proinflammatory molecules involved with the development of atherosclerotic plaques. Herein, we describe the role of thrombin inhibition with anti‐thrombin nanoparticles in limiting plaque deposition and reducing inflammation and hypercoagulability in atherosclerosis.

Methods

ApoE−/− mice fed a Western Diet for 4 months, receiving three intravenous treatment doses per week for the penultimate 4 weeks of Western Diet feeding. Anti‐thrombin nanoparticles were used as the treatment consisting of perfluorocarbon nanoparticles conjugated to multiple copies of the direct thrombin inhibitor D‐phenylalanyl‐L‐prolyl‐L‐arginyl‐chloromethyl ketone (PPACK‐NP). Following 1 month of treatment, aortas were removed and the aortic arch was stained en face for deposited plaques using Sudan IV. Plaque burden in the aortic arch was quantified using ImageJ. Endothelial barrier disruption was quantified by measuring the deposition of semipermeant nanoparticles after 2 hours of circulation; and vessel procoagulant activity measured by time to carotid artery occlusion after dye‐laser injury.

Results

Treatment of ApoE−/− mice for one month with PPACK‐NP resulted in a 22.5% overall decrease (Fig. A) in aortic arch plaque burden compared to mice treated with saline (p = 0.03, Fig. B) in the face of no significant reduction in serum cholesterol (Fig. C). By immunofluorescence, both tissue factor expression and phosphorylation of NF‐kB p65 were reduced (25.94%, p = 0.027 and 21.62%, p = 0.017 respectively). Endothelial barrier function improved by 33% and procoagulant activity diminished by 46%. The activated partial thromboplastin time (APTT) normalized within ~30 minutes after bolus injection of nanoparticles confirming safety of the system with respect bleeding risk.

Conclusion

These results demonstrate the ability of PPACK‐NP treatment to inhibit atherosclerosis in the plaque‐prone aortic arch, restore vascular barrier function, and reduce thrombotic risk without the need for cholesterol lowering or systemic anticoagulation.

Support or Funding Information

Research Support: NIH HL112303
Primary cilia of inner ear hair cells do not respond to mechanical stimuli
Authors: Indzhykulian Artur A. , Delling Markus , Liu Xiaowen , Li Yaqiao , Xie Tiao , Corey David P. , Clapham David E.
Published: November 2023

Hair cells, the sensory cells of the inner ear, carry a bundle of 30–300 specialized actin‐based microvillus‐like projections called stereocilia. Tiny filamentous ‘tip links’ connect the stereocilia and transmit the force produced by sub‐nanometer movements to Ca2+‐permeable mechanoelectrical transduction (MET) channels at the tips of stereocilia—likely encoded by TMC1 and TMC2 genes.

Adjacent to the tallest stereocilia is the kinocilium—a single nonmotile, (9+2) microtubule‐based true cilium. In chicken hair cells, kinocilia are connected to stereocilia by the same proteins as the tip links, raising the question of whether kinocilia also have MET channels. Indeed, TMC1 protein has been reported in kinocilia of mouse hair cells. In developing zebrafish hair cells, moreover, Ca2+ imaging experiments suggest that, prior to the onset of the normal MET, kinocilia mediate a mechanosensitivity elicited by reverse deflections of the hair bundle. Kinocilia and kinocilial links are required for a deflection‐dependent rise in cytoplasmic [Ca2+] in zebrafish.

We used fast Ca2+ imaging to explore the mechanosensitivity of kinocilia on inner and outer hair cells of the mouse cochlea. We developed a transgenic mouse, Arl13b‐mCherry‐GECO1.2, which expresses a genetically‐encoded calcium indicator (GECI) in all primary cilia and, to a lesser extent, in stereocilia. We compared [Ca2+] changes in these two structures (stereocilia and kinocilia) of similar geometry (~5 μm length and 300–600 nm diameter). During hair‐cell development (E14‐P3), we used a swept‐field confocal with millisecond frame time to examine both hair‐cell kinocilia and primary cilia of nearby supporting cells (a negative control). With fast acquisition we could distinguish Ca2+ entry directly into cilia from Ca2+ that back‐diffused from cytoplasm. The fused mCherry and GECO fluorophores allowed ratiometric Ca2+ measurement. We observed robust deflection‐induced Ca2+ influx in stereocilia, but not in kinocilia, consistent with earlier measurements.

Because kinocilia lack Ca2+ influx with motion, we went on to measure responses to flow in primary cilia in tissues upon which this hypothesis has been based: in cultured kidney epithelial cells, kidney thick ascending tubules, embryonic node crown cells, mouse embryonic fibroblasts, and MLOY‐4 and OCY454 osteocyte‐like cell lines. We found a complete lack of mechanically induced calcium increases in these primary cilia. Rapid Ca2+ influx specifically into cilia was not observed with physiological—or even superphysiological—levels of fluid flow in any of these cells, suggesting that primary cilia do not respond to mechanical forces via calcium signaling. We conclude that the Ca2+‐responsive mechanosensor hypothesis of primary cilia should be abandoned.

Support or Funding Information

DPC: NIH 5R01 DC000304 and HHMI; DEC: HHMI.
Induction of Endothelium‐dependent Constriction of Large Arteries in Endotoxemic Hypotensive Rats
Authors: TSENG TZU‐LING , CHEN MEI‐FANG , LIU CHIN‐HUNG , PANG CHENG‐YOONG , HSU YUNG‐HSIANG , LEE Tony J.F.
Published: November 2023

Effective managements of refractory hypotension to vasoconstrictors in severe sepsis are limited. A new strategy to ameliorate endotoxemic hypotension by inducing endothelium‐dependent constriction of endotoxemic large arteries was examined. Rat endotoxemia was induced by lipopolysaccharide (LPS, 10 mg/kg, iv). Changes of the hemodynamics were measured by in vivo experimentation, reactivity of isolated mesenteric arteries by blood‐vessel myography, and expression of proteins/enzymes by immunohistochemistry, biochemistry, and molecular biology. Six hours after LPS‐challenge, the diminished mean arterial pressure (MAP) was promptly reversed/normalized upon oroxylin‐A (OroA, a flavonoid from Scutellariae radix) injection (iv/ip). In isolated LPS‐treated but not normal mesenteric arteries, OroA (1–10 μM) induced endothelium‐dependent, sustained constriction. This vasoconstriction was blocked by endothelin‐1 (ET‐1)‐receptor antagonists, while OroA further enhanced LPS‐induced expression of endothelin‐converting enzyme (ECE), ET‐1 mRNA and proteins, and ET‐1 release, suggesting that OroA‐induced vasoconstriction was mediated by endothelial ET‐1. Also, OroA enhanced ROCK‐phosphorylation and reversed LPS‐induced suppression of RhoA‐activities in smooth muscle cells exclusively in arteries with endothelium. The activated muscle ROCK‐phosphorylation was blocked by ET‐1‐receptor antagonists and ROCK inhibitors. Moreover, OroA‐post‐treatment suppressed, via inhibiting NF‐κB activation, the inducible‐NOS expression and circulating NO. These results indicated that endothelial ET‐1 in endotoxemic mesenteric arteries, upregulated by LPS and released by OroA, induced prompt‐and‐sustained vasoconstriction via activation of muscle RhoA/ROCK‐pathway. In late endotoxemia, OroA‐induced vasoconstriction was caused partly by OroA‐induced decrease of circulating NO. The endothelium‐dependent constriction of large arteries induced by OroA supports its prompt reversal of diminished MAP. It is concluded that activation of endothelium‐dependent constriction of large resistance arteries and suppression of systemic inflammation offer new strategies for acute management of endotoxemic‐hypotensive shock.

Support or Funding Information

(Supported by MOST, Tzu Chi Foundation, & Tzu Chi University)
Cardioprotective effects of mitochondria‐targeted peptide SS‐20 in two different models of rat ischemia/reperfusion
Authors: Dai Wangde , Cheung Elissa , Alleman Rick J. , Brown David A. , Kloner Robert A.
Published: November 2023

This study sought to determine effects of SS20 on myocardial infarct size in two different models of ischemia/reperfusion. For in vivo studies, Anesthetized Sprague Dawley rats were subjected to 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. Rats were randomized to control group (received saline infusion), low dose SS‐20 group (0.3 mg/kg/hour) and high dose SS‐20 group (3 mg/kg/hour) (n=15 rats in each group). Saline or SS‐20 were delivered into the jugular vein starting 5 minutes after coronary artery occlusion and were continued for one hour post coronary artery reperfusion. Body temperature, heart rate and blood pressure were monitored during the procedure. At the end of 3 hours reperfusion, ischemia risk area, no‐reflow area, and infarct size were measured. The ischemia risk area were similar among the saline (49.5 ± 2.3%), low dose SS‐20 (48.6 ± 2.1%), and high dose SS‐20 groups (48.7 ± 3.0%). Treatment with SS‐20 significantly reduced infarct size, which was expressed as % of risk area, in low dose (62.1 ± 4.4%) and high dose (64.0 ± 4.9%) compared with saline treatment (77.6 ± 2.6%, p=0.001 for both doses). There was no difference in infarct size between low and high dose SS‐20 treatment. The no‐reflow areas were comparable among the saline (23.9 ± 1.7%), low dose SS‐20 (23.7 ± 2.8%), and high dose groups (25.0 ± 2.1%). Body temperature, heart rate and blood pressure were comparable among the 3 groups at baseline, during ischemia, and at the end of 3 hours of reperfusion. In separate in vitro studies, isolated rat hearts were exposed to 20 minutes global ischemia, followed by SS‐20 administration (1μM) throughout the 2h reperfusion. Infarct size was reduced from 44 ± 3% in control group (n=11) to 17 ± 3% in the SS‐20 treatment group (n=5). There were no benefits of SS‐20 on recovery of left ventricular developed pressure, coronary flow, or maximal rates of contraction/relaxation. In summary, SS‐20 significantly reduced infarct size in two different models of myocardial injury, but did not affect hemodynamics or no‐reflow area in rat heart.

Support or Funding Information

Grant support by Stealth BioTherapeutics Inc.
Occludin Degradation Mediated by Autophagy Leads to Blood‐Brain Barrier Disruption in Ischemic Stroke
Authors: Kim Kyeong‐A , Kim Eun‐Sun , Noh Dabi , Kim Eunji , Baek Seung‐Hoon , Bae Ok‐Nam
Published: November 2023

The integrity of blood‐brain barrier (BBB), which shows selective permeability for substances to brain, is significantly damaged under ischemic stroke amplifying ischemic neuronal damage. There have been attempts to identify the exact mechanism of ischemic BBB disruption to minimize brain damage. Autophagy is a catabolic process which involves degradation and recycling of damaged or unnecessary organelles, but excessive autophagy can induce cell damage and death under pathological conditions such as ischemia. Here, we evaluated if autophagy plays a key role in BBB dysfunction under ischemic stroke. In vitro BBB model of bEnd.3 cells were exposed to oxygen‐glucose deprivation (OGD), an in vitro ischemic condition. After exposure to OGD for 18 hours, cell viability was significantly decreased and cellular permeability was impaired. The conversion of LC3‐I to LC3‐II and puncta of LC3 in bEnd.3 were increased, demonstrating that autophagy is induced under ischemic condition. Modulation of autophagy by 3‐methyladenine, an autophagy inhibitor, reversed the conversion of LC3, and restored the impairment of cell permeability induced by OGD. The protein level of occludin, a tight junction protein in BBB, was significantly decreased following OGD exposure, and reversed by 3‐MA. Also, immunofluorescence showed occludin was dispersed from cell membrane and co‐localized with LC3 puncta in bEnd.3 after OGD. Our findings showed that induction of autophagy might contribute to increased permeability through occludin degradation in brain endothelial cells under ischemia, providing a new mechanism of BBB disruption in ischemic stroke.

Support or Funding Information

This work was supported by grants from the Korean government (NRF‐2014R1A1A2058459 from the Ministry of Education and HI14C2284 from the Ministry of Health and Welfare).
Endothelial nitric oxide (eNOS)‐dependent and ‐ independent effects of hypoxia on vascular repair‐relevant functions of human CD34+ cells
Authors: Joshi Shrinidh , Jarajapu Yagna
Published: November 2023

CD34+ hematopoietic stem cells are now known for their cardiovascular regenerative functions, and provide a breakthrough approach for the treatment of ischemic cardiovascular diseases. Vasorepairative functions of these cells are known to be potentiated by exposure to hypoxia. Previous studies have demonstrated that nitric oxide (NO) derived from eNOS plays an important role in the reparative functions of CD34+ cells. The current study evaluated the role of NO in the hypoxic potentiation of proliferation and migration, which are key functions that determine vasorepairative potential of these cells. Peripheral blood was obtained from healthy individuals (n=24) and mononuclear cells (MNCs) were separated by Ficoll centrifugation. MNCs were enriched for lineage negative cells, which were further enriched for CD34+ cells by immunomagnetic selection. Hypoxic exposure to hypoxia (1% oxygen) was accomplished by using a hypoxia chamber (Biospherix, Inc.). Intracellular NO levels were detected by DAF‐FM flow cytometry with and without the following treatments: Nω‐nitro‐l‐arginine‐methyl ester (L‐NAME), stromal derived factor‐1a (SDF) or vascular endothelial growth factor (VEGF). Effects of SDF or VEGF on proliferation and migration of CD34+ cells were evaluated under normoxic or hypoxic conditions. Hypoxic exposure increased intracellular NO levels (P<0.01, n=8) that were not affected by L‐NAME (300 μM) pretreatment (n=8). Basal proliferation and migration were enhanced (P<0.05, n=6) by hypoxic exposure that were resistant to L‐NAME (n=6). Proliferation and migration to either SDF or VEGF were potentiated by hypoxia (P<0.05, n=6), which was partly antagonized by L‐NAME (n=6). These findings suggest that hypoxia induces NO generation independent of eNOS, which potentiates proliferation and migration in CD34+ cells. Stimulatory effects of SDF or VEGF are at least in part mediated by eNOS activation in normoxic and hypoxic conditions.

Support or Funding Information

This work is partly supported by American Heart Association, and the Core Biology Facility at North Dakota State University, that was made possible by NIGMS (P30 GM103332‐01).
PARTICIPATION OF NO/GC/cGMP/K+ CHANNELS IN THE RELATATION INDUCED BY Crotalus durissus cascavella IN MESENTERIC ARTERY
Authors: de Jesus Rafael Leonne Cruz , Santos Suelen Silva , Simões Leticia Oliveira , Vasconcelos Walma , Veras Robson , Casais e Silva Luciana Lyra , Silvia Darizy Flavia
Published: November 2023

Introduction

The studies with toxins from snakes poison have shown promising biological effects, especially those regarding to cardiovascular system, such as hypotensive and vasodilators.

Aims

To investigate the vascular effect of Crotalus durissus cascavella poison (CDC), a high prevalence species in the state of Bahia ‐ Brazil, was used rat superior mesenteric artery.

Methods

Wistar rats (12–16 weeks/250–300g) were euthanized in CO2 camera and the mesenteric artery were isolated and free from connective tissue and cut into rings that was kept in Tyrode solution at 37°C carbonated with 95% of O2 and 5% of CO2. The amperometry technic was used to evaluate NO production in isolated aortic endothelial cells. The significant values was p<0.05.

Results

Cumulative administration of the CDC (0.001 – 30μg/mL) relaxed phenylephrine‐induced contractions in a concentration‐dependent manner (Emax = 47.9 ± 5.0%; n=8) in the endothelial vascular presence, this effect was practically abolished in the endothelial vascular absence (Emax= 5.8 ± 2.4%; n=5; *** p<0.001). The vasorelaxant effect induced by CDC in KCl 80mM‐induced contraction was abolished (Emax= 6.4 ± 0.9%; n=5; *** p<0.001. Pre‐treatment with Indomethacin (10μM), L‐NAME (100μM), and ODQ (10μM) significantly attenuated the relaxation effect induced by CDC (Emax = 29.1 ± 7.1%, n = 6, Emax = 17.5 ± 2.2%, n = 6, Emax = 11.2 ± 3.5%, n = 6, respectively). Additionally, in studies with isolated aortic endothelial cells demonstrated that CDC in concentration of 10 and 90 μg/mL induced significant increase in NO production. In mesenteric rings pre incubated with KCl 20mM, to evaluate K+ channels participation, the vasorelaxant effect induced by CDC was significantly reduced (Emax = 21.3 ± 8%, n = 7). Similar results was observed in the presence of Iberiotoxin (100nM), BaCl2 (30μM) and 4‐aminopiridin (1mM) [(Emax = 13.0 ± 3 %, n= 5, Emax = 21.7 ± 8 %, n= 5 e Emax = 9.5 ± 1.3, %, n=5, respectively).

Conclusion

Those results suggest that the CDC induces vasorelaxant effects in isolated superior mesenteric artery rings from normotensive rats in an endothelium‐dependent way, possibly trough endothelium‐derived relaxing factors, especially the NO, resulting in guanylyl cyclase activation, followed by GMPc production and probably K+ channels activation (BKca, Kv and Kir). Additionally, prostanoids derivated from cyclooxygenase also are involved in the relaxant effect induced by CDC.

3012 ‐ ASPET Cardiovascular Pharmacology – General/Other

Support or Funding Information

Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Stimulation of the Immune System by Biophotonics Methods For Hepatitis B and Syphilis in an Inner City High Risk Population
Authors: Newton David , Einstein George P , Tulp Orien L
Published: November 2023

Both Hepatitis B and Syphilis are sexually transmitted diseases of growing concerns in the U.S.A and globally. Syphilis affects over 12 million people in developing countries and between 700,000 and 1.6 million pregnancies a year. Hepatitis B is a main cause of acute and chronic liver disease in the United States and globally. Developing trends reveal that high‐risk heterosexual transmission is the leading cause of acute HBV infections in adults. Despite aggressive and innovative approaches to eliminate the prevalence of HBV and Syphilis, the medical community's best conventional approaches have not seen notable success rates. The objective of this study is to replace conventional medical approaches with Biophotonics to stimulate immune viability in patients affected with HBV and Syphilis among high‐risk urban population. The Literature Review will examine the immune viability of Biophotonics as a credible option to conventional medicine, explore the high prevalence of HBV and Syphilis in high risk urban setting; and examine the impact of these diseases and the failure of conventional approaches to fully reverse their prevalence. The participants in this study will include 30 men and women of varying ethnicities who have tested positive for HBV, Syphilis or both. Each participant will be treated with Biophotonics therapy and data will be interpreted via Quantitative Analysis. For effective therapy the G. P. Einstein Integrated Sigma Quanta Immunostimulation Energy Doses has been Applied. The findings will likely influence future therapeutic interventions to these diseases and trigger more research in the use of Biophotonics therapies.

Support or Funding Information

Supported by Institutional Resources of USAT and the Einstein Medical Institute
Heterologous Phosphorylation of the Cannabinoid Type 1 receptor by Angiotensin II in Astrocytes isolated from Spontaneously Hypertensive Rats
Authors: Haspula Dhanush , Clark Michelle
Published: November 2023

Objective

To determine if a mediator of neuroprotection, the Cannabinoid Receptor Type 1 (CB1R), is phosphorylated by Angiotensin II (Ang II) via the Angiotension Type 1 receptor (AT1R) in astrocytes.

Background

AT1R and CB1R have opposing roles in regulating the release of neuroinflammatory cytokines from astrocytes. While the former, a Gq G protein coupled receptor (GPCR), is pro‐inflammatory, the latter is anti‐inflammatory. Activation of Gq GPCRs have been demonstrated to inactivate the CB1R via a key phosphorylation event.

Methods

Astrocytes were isolated from the brainstem and cerebellum of Spontaneously Hypertensive Rats (SHR), a model of hypertension and Attention Deficit Hyperactivity Disorder (ADHD). The cells were treated with 100 Nm Ang II for varying time periods and the extent of phosphorylation was measured using Western blotting and Flow cytometry. To determine the receptor involved, the cells were also pretreated with 10 μM Losartan and 10 μM PD123319, the AT1 and AT2 receptor antagonists respectively.

Results

Basal CB1R was significantly higher in SHR when compared to Wistar. Higher levels were observed in cerebellum. Ang II phosphorylated CB1R in both brain regions of both rat models. This effect was predominantly mediated through the AT1R. The phosphorylation event was initiated earlier and was sustained longer in SHR when compared to wistar.

Conclusion

Higher basal CB1R phosphorylation in SHR as opposed to Wistar is suggestive of a potential diminished protective effect in pathological conditions such as hypertension and ADHD. Also Ang II could be involved in disinhibiting CB1R actions via the AT1R by heterologous desensitization. Temporal and magnitude differences in phosphorylation of CB1R by Ang II, in SHR versus Wistar, are suggestive of a potential therapeutic target to combat neuroinflammation.

Support or Funding Information

NSU HPD: Grant # 335585
Insulin‐Mediated Vasodilation in Cerebral Arteries is Impaired in Obese Rats with Type 2 Diabetes
Authors: Olver T. Dylan , McDonald Matt W , Klakotskaia Diana , Richardson Rachel A , Jasperse Jeffrey A , Yang Hsiao T , Thorne Pam K , Melling C.W. James , Schactman Todd R. , Emter Craig A , Laughlin M. Harold
Published: November 2023

Insulin stimulates resistance artery vasodilation in skeletal muscle and the brain. Type 2 diabetes (T2D) attenuates insulin‐induced vasodilation in skeletal muscle arteries, but whether this adaptation occurs in the cerebrovasculature and is related to impaired cognition remains to be elucidated. This study tested the hypothesis that cognition and insulin‐mediated vasodilation in the cerebrovasculature were impaired in obese rats with type 2 diabetes. Eight week old hyperphagic, Otsuka Long Evans Tokushima Fatty rats were divided into two groups; food controlled (CON, n=8) and free food access (model of T2D, n=8). Cognition was measured monthly using a Barnes maze for 4 months (i.e. at 8, 12, 16 and 20 wk of age) prior to vascular testing. At 20 wk of age, rats underwent a euglycemic hyperinsulinemic clamp, while carotid artery blood flow (BF; perivascular flow probe) and mean arterial pressure (MAP; arterial catheter) were measured. Thereafter, rats were decapitated, their cerebral arteries were isolated, and vasomotor responses (pressurized myography) to graded increases in intraluminal pressure (40–140 mmHg) and exogenous insulin (1–10000 uIU) were assessed in untreated and endothelin‐1 inhibition (BQ123) conditions. Results indicate that cognition improved over time, but was not different between groups at any time point. Carotid BF, MAP and vascular conductance (BF/MAP) were also not different between groups and did not change during the insulin clamp. Prior to and following the development of spontaneous tone, absolute diameter of the cerebral arteries was not different between groups. Vasomotor tone increased similarly in both groups during graded increases in intraluminal pressure (40–140 mmHg; P<0.05). Insulin‐mediated vasodilation (at 80 mmHg pressure) was attenuated in the T2D vs. CON (peak dilation = ~10 % vs. 30%; P<0.05). Endothelin‐1 inhibition restored insulin‐mediated vasodilation in the T2D group. A priori pairwise comparison between the cumulative average percent possible insulin‐mediated vasodilation in the untreated vs. endothelin‐1 inhibition conditions revealed that endothelin‐1 significantly restrained insulin‐mediated vasodilation in the T2D, but not the CON group (P<0.05). Thus, at this stage in development of T2D (~20 wk is the onset in this model), cognition, and cranial flow at rest and during an insulin clamp appear unaffected; even though isolated cerebral arteries exhibit vascular insulin resistance. These data from isolated cerebral arteries support the notion that impaired insulin‐mediated cerebral vasodilation in T2D is the result of enhanced insulin‐stimulated endothelin‐1 production.

Support or Funding Information

College of Veterinary Medicine, University of Missouri‐Columbia
The Effects of Captopril Treatment and Exercise and Blood Pressure, Endothelium Function and Gene Expression of the Renin Angiotensin System in the Spontaneously Hypertensive Rat
Authors: Enyinnia Diamond Enyinnia , Henry Eden , Okoire Nnaemeka , Vaden Deirdre , Keaton Alphonso
Published: November 2023

Angiotensin converting enzyme inhibitors (ACE‐I) have been show to prevent impairment of endothelial cell function in the Spontaneously Hypertensive Rat (SHR). The purpose of this study was to examine the combined effects of long‐term exercise training and ACE‐I treatment on blood pressure, vascular reactivity and gene expression of the Renin Angiotensin System (RAS), in the SHR. Three groups of male SHR were studied: 1) untreated SHR; 2) exercise trained (35 cm/s) for 60 minutes/day, for 3 days/week (SHREX); 3) exercised trained + captopril treated (50 mg/kg/day), treatment was initiated at four weeks of age and continued for 16 weeks (CAPEX). N = 8, for all three groups. At 20 weeks of age, systolic blood pressure (via tail cuff) in the untreated SHR was 190±11 mmHg. Exercise treatment in the SHR significantly decrease MAP in the SHREX was 151±7 mmHg). The combination of exercise and oral administration of captopril was associated with a significant decrease in systolic blood pressure (130±3 mmHg) compared to untreated SHR and SHREX. Isolated 3mm aortic ring segments were suspended in tissue chambers for measurement of isometric force. Ring segments were exposed to cumulative concentrations of serotonin (3× 10−9 – 3 × 10−5M). Preliminary data from aortic rings from SHR demonstrated increased responsiveness to serotonin induced contraction in comparison to aortic rings from the SHREX and CAPEX group. Endothelium‐dependent relaxation to acetylcholine was dramatically impaired in the SHR. Harvested tissue samples from the brain, kidney liver and blood will be used to examine gene expression of the renin angiotensin system using Next Generation RNA Sequencing.

Support or Funding Information

Mini Grant Award from the Office of Research and Graduate School, Prairie View A&M University
The Effect of Salbutamol on the Vasculature Differs in Asthmatics As Compared to Healthy Controls
Authors: Moore Linn E , Byers Bradley W , Kapoor Karishma , Stickland Michael K
Published: November 2023

INTRODUCTION

Asthmatics are at increased risk of cardiovascular (CV) disease and inhaled beta(β)2‐agonist use is associated with increased CV events. Short‐acting β2‐agonists acutely affect cardiovascular function among healthy individuals. However, chronic β‐agonist use, often seen in asthma, is associated with β2‐refractoriness within the airways. It is unknown if the vascular responses to a clinically relevant dose of acute β2‐agonist differ between asthmatics and healthy controls.

PURPOSE

To compare the vascular response to an inhaled β2‐agonist between controls and asthmatics.

METHODS

Eight asthmatics and 8 healthy controls matched for age, height and weight were recruited for the study. Average short‐term β2‐agonist usage among the asthmatics was 3.1 times/week for 11.5 years. Blood pressure (BP; mmHg), arterial stiffness (PWV; m/s), microvascular function (VTI; cm/s), and endothelial function (FMD; % baseline/shear rate) were examined before and after inhaled beta agonist (4 x 100 μg Salbutamol), or placebo. The order of test days was randomized, but all testing was conducted at the same time of day following a 12 hour fast. Respiratory medications were withheld for 8 hours prior to each test day.

RESULTS

There were no baseline differences in arterial stiffness between groups. However, arterial stiffness increased in asthmatics but not controls acutely following Salbutamol administration (ΔPWV: +0.62 vs. −0.08 m/s, p<0.05). There was no between‐group difference in endothelial function or microvascular function at baseline; nor following β2‐agonist inhalation. Both groups had increased systolic blood pressure following Salbutamol administration which was not seen with placebo (Placebo: 109 to 108 mmHg, Salbutamol: 108 to 113 mmHg, p<0.05).

CONCLUSION

Asthmatics have an altered acute vascular response to a clinically relevant dose of a short‐acting β2‐agonist, which results in an increase in arterial stiffness in asthmatics which is not seen in controls. The vascular stiffening in response to beta‐agonist use may contribute to the elevated CV risk seen in asthma.

Support or Funding Information

The Lung Association, Alberta & NWT
Vitamin D Receptor Expression is Essential for the Antiangiogenic Activity of Vitamin D
Authors: Jamali Nasim , Sorenson Christine M , Sheibani Nader
Published: November 2023

Vitamin D compounds inhibit the growth of a variety of tumors perhaps through inhibition of angiogenesis. We previously showed that the active form of vitamin D, calcitriol (1, 25‐dihydroxyvitamin D3), is a potent inhibitor of neovascularization. However, the molecular and cellular mechanisms involved remain unknown. Here we determined whether the expression of the vitamin D receptor is essential for the antiangiogenic activity of vitamin D using mice deficient in vitamin D receptor (Vdr −/−). We also determined the impact of VDR deficiency in postnatal development of retinal vasculature. Wild type (Vdr +/+), heterozygous (Vdr +/−), and null (Vdr −/−) mice were subjected to oxygen‐induced ischemic retinopathy (OIR). During OIR postnatal day 7 (P7) mice are exposed to 75% oxygen for 5 days (P12) and then brought to room air for 5 days (P17). Maximum neovascularization occurs from P12‐P17 and degree of neovascularization is assessed at P17. To determine antiangiogenic activity of vitamin D animals received a daily intraperitoneal injection of calcitriol from P12‐P17. We showed that VDR expression is essential for inhibition of neovascularization, which was most evident in the female mice. Calcitriol failed to inhibit neovascularization in Vdr −/− mice compare to Vdr +/+ or Vdr +/− mice. Furthermore Vdr −/−, unlike the Vdr +/+, continued to gain weight following calcitriol treatment. We also observed a significant effect on the density of retinal vasculature and the number and structure of major retinal arteries during normal development. Mechanistically, we showed that retinal pericytes (PC) express significantly higher level of VDR compared with retinal endothelial cells. Calcitriol also attenuated the proliferation and migration of PC, and caused G0/G1 arrest without affecting apoptosis. Together, our results demonstrate an important role for VDR expression and inhibition of neovascularization perhaps through attenuation of PC proliferation and migration.

Support or Funding Information

P30 EY016665, RPB, and RRF.
Preeclampsia in the rat: validation of the deoxycorticosterone (DOCA)‐salt model
Authors: Hanson Andrea E , Stallone John N
Published: November 2023

Preeclampsia (PE) is a multi‐system disorder that typically occurs during the second and third trimesters of pregnancy. PE complicates 7–10% of all pregnancies worldwide and is a major cause of fetal and maternal morbidity and mortality. The hallmarks of PE include pregnancy induced‐hypertension (PIH), proteinuria, reduction in renal function, intrauterine growth restriction (IUGR) of the fetus, chronic immune activation, and multi‐organ dysfunction. The pathology of PE is not well understood. In the present study, DOCA‐salt treatment was validated as a model of PE in the female (F) Sprague‐Dawley rat. Normal pregnant (NP) and DOCA‐salt treated pregnant (DSP) F rats were studied, using time release DOCA‐impregnated silicone rubber implants (200 mg/kg) and saline drinking water (1% NaCl/0.2% KCl) from the onset of pregnancy until term. At term (day 18–20 of pregnancy), systolic blood pressure (tail cuff method) was elevated in DSP (146 ± 2 mmHg) vs. NP (115 ± 1 mmHg). In DSP, total urinary protein excretion was measurably higher in DSP (29.67 ± 11 mg/day) compared to NP (14.25 ± 10 mg/day). In addition, fetal weight was lower in DSP (2.52 ± 0.09 g) than in NP (4.05 ± 0.34 g), and maternal body weight was higher in NP (388 ± 5 g) compared to DSP (355 ± 25 g). These data suggest that DOCA‐salt treatment creates an accurate model of PIH and PE in the F rat.

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(State of Texas)
Angiotensin II Type 1 Receptor Agonistic Autoantibody Inhibition Improves Maternal Outcomes in Reduce Uterine Perfusion Pressure Rats
Authors: Cunningham Mark W. , Castillo Javier , Spradley Frank , Faulkner Jessica , Ibrahim Tarek , Elfarra Jamil , LaMarca Babbette
Published: November 2023

Background

Women with preeclampsia have hypertension, placental ischemia, intrauterine growth restriction, reduced glomerular filtration rate (GFR), elevated sFlt, preproendothelin (PPE), and agonistic autoantibodies to the Angiotensin II type 1 receptor (AT1‐AA) during pregnancy. The reduce uterine perfusion pressure (RUPP) rat model of placental ischemia during pregnancy, also displays elevated AT1‐AAs, blood pressure, sFlt, PPE, and a reduced GFR along with fetus and placenta demise. Due to the robust elevation of AT1‐ AAs in the pathophysiology of preeclampsia, we hypothesis that administration of a specific 7 amino acid peptide sequence (7AA) to inhibit the AT1‐AA, will improve blood pressure, sFlt, preproendothelin, GFR, and pregnancy outcomes in RUPP rats.

Methods

Day 14 of gestation rats receive the RUPP procedure with or without the AT1‐AA inhibitory peptide (2ug/ml saline) via mini‐osmotic pumps, infusing 7AA peptide for 6 days. On day 19 of gestation, conscious blood pressure (MAP) and GFR were determined, pups and placentas were counted and weighed, and other tissues were collected. All rats were age matched to control normal pregnant (NP) rats.

Results

MAP was elevated in RUPP rats (n=28) vs. NP rats (n=10) (121±1 mmHg vs .98±3 mmHg, p<0.05; respectively). The rise in blood pressure was returned to NP baseline blood pressures in RUPP rats treated with the 7AA (n= 15; 104±3 mmHg, p<0.05). Pup weight, number of pups, and placental weight was decreased in RUPP rats vs. NP rats and remained unchanged with RUPP + 7AA rats. Circulating sFlt1 (176±37 vs. 77±15pg/ml, p<0.05) was elevated in RUPP rats vs. NP rats and was normalized in RUPP + 7AA rats (86±9 pg/ml, p<0.05). There was a 4 fold increase in PPE in RUPP (n=7) vs. NP (n=6) rats and a 2 fold decreased in PPE between RUPP + 7AA (n=7) vs. RUPP rats (p<0.05). GFR was improved in RUPP + 7AA (n=2) vs RUPP rats (n=3) (1.4±0.12 vs. 1.1±0.05 ml/min/100g BW).

Conclusion

AT1‐AA inhibition improves the maternal blood pressure, GFR, and secretion of anti‐angiogenic and vasoconstrictor factors in RUPP rats, and thus maybe a potential therapy for preeclamptic patients.

Support or Funding Information

Research Supported by T32HL105324 and RO1HD067541
Increased Myogenic Tone and Underlying [Ca2+]i and Arterial Stiffness in Mesenteric Microvessels of Hypertensive Pregnant Rats
Authors: Mazzuca Marc Q. , Buyukcelebi Kadir , Ren Zongli , Zhu Minglin , Khalil Raouf A.
Published: November 2023

Preeclampsia is a pregnancy‐related hypertensive disorder (HTN‐Preg) with unclear mechanisms. We have previously shown enhanced vasoconstriction and microvascular reactivity to external KCI‐induced membrane depolarization and endothelin‐1 in HTN‐Preg rats with reduced uterine perfusion pressure (RUPP). However, whether HTN‐Preg involves changes in the intrinsic myogenic responses of systemic microvessels is less clear. In Norm‐Preg and RUPP rats on day 19 of pregnancy, BP was recorded and mesenteric microvessels were isolated and mounted on a pressure myograph system for measurement of myogenic tone, arterial stiffness and simultaneous changes in [Ca2+]i (fura‐2 340/380 ratio). Microvessels were incubated in Ca2+ containing Krebs or 0 Ca2++EGTA Krebs and pressurized from 0–110 mmHg in 10 mmHg increments for measurement of active or passive myogenic responses, respectively. The myogenic response was expressed as % change in vessel diameter from initial diameter at 10 mmHg. BP was higher in RUPP (133±3 mmHg) than Norm‐Preg rats (102±2 mmHg). In Ca2+ containing Krebs, active myogenic tone was greater in microvessels of RUPP (34.5±2.6%) than Norm‐Preg rats (26.5±2.9%), and was associated with greater increases in [Ca2+]i in RUPP (0.90±0.01) than Norm‐Preg rats (0.86±0.01). In 0 Ca2++EGTA Krebs, the passive myogenic response was also increased in microvessels of RUPP (36.2±1.1%) vs. Norm‐Preg rats (19.3±1.2%), with no underlying changes in [Ca2+]i, suggesting changes in the structure and mechanics of the arterial wall. The active and passive stress‐strain relationship for microvessels of RUPP rats was shifted to the left compared to Norm‐Preg rats, suggesting increases in arterial wall stiffness. Thus, the increase in BP in placental ischemia model of HTN‐Preg is associated with increased myogenic tone and arterial stiffness in mesenteric microvessels. These results suggest that intrinsic alterations in vascular smooth muscle Ca2+ signaling as well as structural changes in the microvascular wall architecture could play a role in the increased vasoconstriction and BP in HTN‐Preg.

Support or Funding Information

This work was supported by grants from the National Heart, Lung, and Blood Institute (HL‐65998, HL‐111775). M.Q. Mazzuca is a recipient of American Heart Association Postdoctoral Fellowship (Founders Affiliate) Award no. 14POST18150007.