Thromboxane A2 and TP receptors: A trail of research, well traveled

This article was written is response to a request from the editor-in-chief as a result of my receiving the Governor’s Award in Scientific Awareness. My goal is to provide a glimpse into a 40 plus year research career that was predominately focused on the role of thromboxane A2 and its receptor (TP) in physiologic and pathophysiologic processes in the cardiovascular system. It also enumerates the lessons learned along the way that may lead to a productive research career. Those lessons were; Perseverance, Think out of the box, Challenge the dogma, Take risks, Failure is not a bad thing, Focus, Select a good mentor(s), Choose your collaborators and colleagues wisely (team science), When you start an experiment-finish it (analyze all the data), Take time for your family

Thromboxane and Stable Prostaglandin Endoperoxide Analogs Stimulate Water Permeability in the Toad Urinary Bladder

The effects of thromboxane B2 and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9α - 1 lα - (epoxymethano)prosta - 5Z,13E – dienoic acid (U44069) and (15Z)-hydroxy-1 lα,9α-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A2 synthesis, thromboxane B2 stimulated water flow in a dose-dependent manner. U44069 (1 µM) stimulated water flow from 3.6±0.8 to 12.4±1.2 mg/min per 10 cm2 hemibladder surface area, while U46619 (1 µM) stimulated water flow from 2.8± 1.0 to 21.8±2.0 mg/min per 10 cm2. The prostaglandin endoperoxide/thromboxane A2 antagonist trans-13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B2- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 µM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P \u3c 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A2-like compounds stimulate water flow in the toad urinary bladder

交感神経β受容体遮断薬propranololの降圧機序に関する研究 : 血管拡張性prostaglandinの役割を中心に

β-adrenergic receptor antagonists are widely used to treat the patients with hypertension. The mechanism of the antihypertensive effect of these drugs was thought to be associated with 1) negative cardiac inotropic and chronotropic action, 2) suppression of renin secretion, and 3) effects on the central nervous system. However, the precise mechanism is still unknown. Long term therapy with β-adrenergic receptor antagonists is also associated with a reduction in the peripheral vascular resistance. Indomethacin and other non-steroidal antiinflammatory drugs have been shown to blunt the antihypertensive effect of propranolol and other β-adrenergic receptor antagonists in patients and laboratory animals. This study was, therefore, designed to determine the effects of DL-, D- or L-propranolol on the synthesis of the vasodilatory prostaglandin, PGI2 and PGE2 in the aorta, renal medulla, and the renal cortex of spontaneously hypertensive rats (SHR). DL-Propranolol and L-propranolol significantly lowered blood pressures from 148±9/113±5mmHg to 112±3/80±3 and from 133±4/100±2mmHg to 121±3/81±3 mmHg, respectively. Comparable treatment of SHR with D-propranolol revealed no lowering effect. Basal immunoreactive 6-keto PGF1α and PGE2 production by isolated thoracic aorta, renal medulla, and renal cortex was measured in the samples with an incubation time of 60 to 90 minutes and showed no difference in the vehicle compared to the DL-propranolol treated rats. Norepinephrine (1 μM)-stimulated synthesis of 6-keto PGF1α and PGE2 in the aorta was significantly enhanced in the DL-propranolol treated group compared to the vehicle treated group. Aortic 6-keto PGF1α, and PGE2 synthesis stimulated by norepinephrine in the L-propranolol treated rats was also significantly higher than that observed in the vehicle and D-propranolol treated groups. DL-propranolol treatment did not alter norepinephrine-stimulated renal cortical or medullary 6-keto PGF1α and PGE2 production. Bradykinin- and angiotensin II- augmented aortic 6-keto PGF1α along with PGE2 production in the chronic treatment with L-propranolol was significantly higher than that in the D-propranolol treated group. However, arachidonic acid-stimulated aortic 6-keto PGF1α and PGE2 production in the chronic treatment with DL-propranolol group was not different from that of the vehicle treated group. Acute in vitro exposure of thoracic aortas from SHR to D-, or L-propranolol (100ng/ml) did not ?augment aortic 6-keto PGF1α and PGE 2 synthesis compared to the vehicle. These data suggest that 1) the antihypertensive effect of propranolol is stereoselective, 2) norepinephrine-, bradykinin-, and angiotensin II-stimulated aortic vasodilatory prostaglandin synthesis are stereoselective, 3) the antihypertensive effect of propranolol may be related to the enhanced synthesis of vasodilatory prostaglandin, PGI2 or PGE2, in the vascular tissue, and 4) chronic DL-propranolol treatment may modulate arachidonic acid metabolism by changing either phospholipase A2 activity or the membrane phospholipids metabolism

Circulating extracellular vesicles are associated with the clinical outcomes of sepsis

IntroductionSepsis is associated with endothelial cell (EC) dysfunction, increased vascular permeability and organ injury, which may lead to mortality, acute respiratory distress syndrome (ARDS) and acute renal failure (ARF). There are no reliable biomarkers to predict these sepsis complications at present. Recent evidence suggests that circulating extracellular vesicles (EVs) and their content caspase-1 and miR-126 may play a critical role in modulating vascular injury in sepsis; however, the association between circulating EVs and sepsis outcomes remains largely unknown.MethodsWe obtained plasma samples from septic patients (n=96) within 24 hours of hospital admission and from healthy controls (n=45). Total, monocyte- or EC-derived EVs were isolated from the plasma samples. Transendothelial electrical resistance (TEER) was used as an indicator of EC dysfunction. Caspase-1 activity in EVs was detected and their association with sepsis outcomes including mortality, ARDS and ARF was analyzed. In another set of experiments, total EVs were isolated from plasma samples of 12 septic patients and 12 non-septic critical illness controls on days 1, and 3 after hospital admission. RNAs were isolated from these EVs and Next-generation sequencing was performed. The association between miR-126 levels and sepsis outcomes such as mortality, ARDS and ARF was analyzed.ResultsSeptic patients with circulating EVs that induced EC injury (lower transendothelial electrical resistance) were more likely to experience ARDS (p<0.05). Higher caspase-1 activity in total EVs, monocyte- or EC-derived EVs was significantly associated with the development of ARDS (p<0.05). MiR-126-3p levels in EC EVs were significantly decreased in ARDS patients compared with healthy controls (p<0.05). Moreover, a decline in miR-126-5p levels from day 1 to day 3 was associated with increased mortality, ARDS and ARF; while decline in miR-126-3p levels from day 1 to day 3 was associated with ARDS development.ConclusionsEnhanced caspase-1 activity and declining miR-126 levels in circulating EVs are associated with sepsis-related organ failure and mortality. Extracellular vesicular contents may serve as novel prognostic biomarkers and/or targets for future therapeutic approaches in sepsis

Alterations in macrophage G proteins are associated with endotoxin tolerance

AbstractPrevious studies have suggested that endotoxin tolerance induces macrophage desensitization to endotoxin through altered guanine nucleotide regulatory (G) protein function. In the present study the binding characteristics of the nonhydrolyzable GTP analogue GTPγ[35S] to macrophage membranes from endotoxin tolerant and control rats were determined. Membranes were prepared from peritoneal macrophages harvested from rats 72 h after two sequential daily doses of vehicle or Salmonella enteritidis endotoxin (100 μg/kg on day 1 and 500 μg/kg on day 2). GTPγ[35S] bound to a single class of sites that were saturable and displaceable in control and endotoxin tolerant macrophage membranes. The maximum specific binding of GTPγ[35S] was significantly (P < 0.01) decreased in membranes from tolerant rats compared to control (Bmax = 39 ± 7 pmol/mg protein in control vs. 11 ± 2 pmol/mg protein in endotoxin tolerant; n = 5). There were no significant differences in the Kd values. To determine whether the reduced GTPγS binding was due to decreases in G proteins, macrophage membrane G protein content was determined by western blotting with specific antisera to Gi1,2 α, Gi3α, Gs α, and the β subunit of G. Scanning densitometric analysis demonstrated differential decreases in tolerant macrophage membrane G proteins. Gi3α was reduced the most to 48 ± 8% of controls (n = 3), and this reduction was significant compared to those of other G proteins. Gi1,2α and Gβ were reduced to 73 ± 5% (n = 3) and 65 ± 4% (n = 3) of control values, respectively. Gs α) (L) and Gs α(H) were reduced to 61 ± 5% (n = 3) and 68 ± 3% (n = 3) of control, respectively. These results demonstrate that endotoxin tolerant macrophages exhibit decreased membrane GTP binding capacity and differential reductions in the content of specific G proteins. The cellular mechanisms leading to such alterations in G proteins and their functional significance in the acquisition of endotoxin tolerance merit further investigation

FLI1 in PBMCs contributes to elevated inflammation in combat-related posttraumatic stress disorder

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition with significant public health implications that arise following exposure to traumatic events. Recent studies highlight the involvement of immune dysregulation in PTSD, characterized by elevated inflammatory markers. However, the precise mechanisms underlying this immune imbalance remain unclear. Previous research has implicated friend leukemia virus integration 1 (FLI1), an erythroblast transformation-specific (ETS) transcription factor, in inflammatory responses in sepsis and Alzheimer’s disease. Elevated FLI1 levels in peripheral blood mononuclear cells (PBMCs) have been linked to lupus severity. Yet, FLI1’s role in PTSD-related inflammation remains unexplored. In our study, PBMCs were collected from Veterans with and without PTSD. We found significantly increased FLI1 expression in PBMCs from PTSD-afflicted Veterans, particularly in CD4+ T cells, with no notable changes in CD8+ T cells. Stimulation with LPS led to heightened FLI1 expression and elevated levels of inflammatory cytokines IL-6 and IFNγ in PTSD PBMCs compared to controls. Knockdown of FLI1 using Gapmers in PTSD PBMCs resulted in a marked reduction in inflammatory cytokine levels, restoring them to control group levels. Additionally, co-culturing PBMCs from both control and PTSD Veterans with the human brain microglia cell line HMC3 revealed increased inflammatory mediator levels in HMC3. Remarkably, HMC3 cells co-cultured with PTSD PBMCs treated with FLI1 Gapmers exhibited significantly lower inflammatory mediator levels compared to control Gapmer-treated PTSD PBMCs. These findings suggest that suppressing FLI1 may rebalance immune activity in PBMCs and mitigate microglial activation in the brain. Such insights could provide novel therapeutic strategies for PTSD

Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by Gαi proteins

AbstractHeterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Gαi2 (−/−) or Gαi1/3 (−/−) mice were investigated. LPS- or SA-induced production of TNFα, IL-6, IFNγ, IL-12, IL-17, GM-CSF, MIP-1α, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p<0.05) in splenocytes harvested from Gαi2(−/−) mice compared with WT mice. The effect of Gαi protein depletion was remarkably isoform specific. In splenocytes from Gαi1/3 (−/−) mice relative to WT mice, SA-induced IL-6, IFNγ, GM-CSF, and IP-10 levels were decreased (59% to 86%, p<0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Gαi2 (−/−) and Gαi1/3 (−/−) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Gαi2 (−/−) and Gαi1/3 (−/−) mice relative to WT mice. The disparate response of splenocytes from the Gαi2 (−/−) relative to Gαi1/3 (−/−) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that Gi2 and Gi1/3 proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli

Characterization of Human Peripheral Blood Monocyte Thromboxane A2 Receptors1

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Thromboxane and Stable Prostaglandin Endoperoxide Analogs Stimulate Water Permeability in the Toad Urinary Bladder

The effects of thromboxane B(2) and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9α - 11α - (epoxymethano)prosta - 5Z,13E - dienoic acid (U44069) and (15Z)-hydroxy -11α,9α-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A(2) synthesis, thromboxane B(2) stimulated water flow in a dose-dependent manner. U44069 (1 μM) stimulated water flow from 3.6±0.8 to 12.4±1.2 mg/min per 10 cm(2) hemibladder surface area, while U46619 (1 μM) stimulated water flow from 2.8±1.0 to 21.8±2.0 mg/min per 10 cm(2). The prostaglandin endoperoxide/thromboxane A(2) antagonist trans- 13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B(2)- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 μM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P < 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A(2)-like compounds stimulate water flow in the toad urinary bladder