Changes in circulating exosome molecular profiles following surgery/(chemo)radiotherapy: early detection of response in head and neck cancer patients.

BACKGROUND: Head and neck cancers (HNSCC) are highly immunosuppressive. Plasma-derived exosomes of HNSCC patients carry immunomodulatory molecules, and their cargo correlates with clinical parameters. Here, we evaluated the exosomal molecular profile for early detection of treatment failure in locally advanced HNSCC patients treated with conventional therapy. METHODS: Plasma from 17 HNSCC patients was collected before, during, and after treatment by surgery with adjuvant (chemo)radiation and at recurrence. Exosomes were isolated by size-exclusion chromatography. Total exosomal protein (TEP) was used to estimate exosome load and on-bead flow cytometry to evaluate relative fluorescence intensity (RFI) of tumour-associated and immunoregulatory proteins on exosomes. Exosomal effects on the activity of and adenosine production by T cells was assessed by flow cytometry and mass spectrometry. RESULTS: TEP and the ratio of tumour-/immune-cell-derived exosomes varied during and after therapy with an overall decrease in the tumour-free follow-up but an increase at recurrence. RFI values of immunoregulatory proteins on exosomes, their ability for T cell inhibition and adenosine production changed during and after therapy. PD-L1 was the earliest discriminator for treatment failure and disease-free survival. CONCLUSIONS: Monitoring of plasma exosomes during therapy represents a promising opportunity for early detection of treatment failure and risk stratification to delay/avoid recurrence.

Adenosine production in mesenchymal stromal cells in relation to their developmental status.

BACKGROUND: Mesenchymal stromal cells (MSC) are multipotent progenitor cells found in the tumor microenvironment. They have an innate and regulatory immune activity, and they are able to produce immunosuppressive adenosine (ADO) via their ectonucleotidases CD39 and CD73. The present study explores ADO metabolism of MSC in relation to their developmental status. METHODS: We analyzed MSC (n = 6), chondrogenic progenitor cells (CPC, n = 8), and chondrocytes (n = 8) for surface markers by flow cytometry. The ability to hydrolyze ATP and to produce ADO was tested by luminescence assays and mass spectrometry. RESULTS: Significant differences in the surface marker expression of MSC, CPC, and chondrocytes were seen. While the expression of CD73 was observed to be the same on all cell types, the expression of the ectonucleotidase CD39 was significantly increased on MSC. Consequently, production of ADO was most abundant in MSC as compared with chondrocytes and CPC. CONCLUSION: Mesenchymal stromal cells are potent producers of ADO and are, therefore, able to increase immunosuppression. As MSC differentiate into chondrocytes, they lose this ability and may take on other functions.

Exosomes in HNSCC plasma as surrogate markers of tumour progression and immune competence.

Exosomes in plasma of head and neck squamous cell carcinoma (HNSCC) patients comprise subsets of vesicles derived from various cells. Recently, we separated CD3(+) from CD3(-) exosomes by immune capture. CD3(-) exosomes were largely tumour-derived (CD44v3+ ). Both subsets carried immunosuppressive proteins and inhibited functions of human immune cells. The role of these subsets in immune cell reprogramming by the tumour was investigated by focusing on the adenosine pathway components. Spontaneous adenosine production by CD3(+) or CD3(-) exosomes was measured by mass spectrometry, as was the production of adenosine by CD4+ CD39+ regulatory T cells (Treg ) co-incubated with these exosomes. The highest level of CD39/CD73 ectoenzymes and of adenosine production was found in CD3(-) exosomes in patients with the stages III/IV HNSCCs). Also, the production of 5'-AMP and purines was significantly higher in Treg co-incubated with CD3(-) than CD3(+) exosomes. Consistently, CD26 and adenosine deaminase (ADA) levels were higher in CD3(+) than CD3(-) exosomes. ADA and CD26 levels in CD3(+) exosomes were significantly higher in patients with early (stages I/II) than advanced (stages III/IV) disease. HNSCC patients receiving and responding to photodynamic therapy had increased ADA levels in CD3(+) exosomes with no increase in CD3(-) exosomes. The opposite roles of CD3(+) ADA+ CD26+ and CD3(-) CD44v3+ adenosine-producing exosomes in early versus advanced HNSCC suggest that, like their parent cells, these exosomes serve as surrogates of immune suppression in cancer.

Phenotypic and functional characteristics of CD39high human regulatory B cells (Breg).

CD39 and CD73 are key enzymes in the adenosine (ADO) pathway. ADO modulates pathophysiological responses of immune cells, including B cells. It has recently emerged that a subpopulation of ADO-producing CD39+CD73+ B cells has regulatory properties. Here, we define the CD39high subset of these cells as the major contributor to the regulatory network operated by human B lymphocytes. Peripheral blood B cells were sorted into CD39neg, CD39inter and CD39high subsets. The phenotype, proliferation and IL-10 secretion by these B cells were studied by flow cytometry. 5'-AMP and ADO levels were measured by mass spectrometry. Agonists or antagonists of A1R, A2AR and A3R were used to study ADO-receptor signaling in B cells. Inhibition of effector T-cell (Teff) activation/proliferation by B cells was assessed in co-cultures. Cytokine production was measured by Luminex. Upon in vitro activation and culture of B cells, the subset of CD39high B cells increased in frequency (p < 0.001). CD39high B cells upregulated CD73 expression, proliferated (approximately 40% of CD39high B cells were Ki-67+ and secreted fold-2 higher IL-10 and ADO levels than CD39neg or CD39inter B cells. CD39high B cells co-cultured with autologous Teff suppressed T-cell activation/proliferation and secreted elevated levels of IL-6 and IL-10. The A1R and A2AR agonists promoted expansion and functions of CD39high B cells. CD39 ectonucleotidase is upregulated in a subset of in vitro-activated B cells which utilize ADO and IL-10 to suppress Teff functions. Proliferation and functions of these CD39high B cells are regulated by A1R- and A2AR-mediated autocrine signaling.

Human CD4+ CD39+ regulatory T cells produce adenosine upon co-expression of surface CD73 or contact with CD73+ exosomes or CD73+ cells.

While murine CD4(+) CD39(+) regulatory T cells (T(reg)) co-express CD73 and hydrolyze exogenous (e) adenosine triphosphate (ATP) to immunosuppressive adenosine (ADO), surface co-expression of CD73 on human circulating CD4(+) CD39(+) T(reg) is rare. Therefore, the ability of human T(reg) to produce and utilize ADO for suppression remains unclear. Using mass spectrometry, we measured nucleoside production by subsets of human CD4(+) CD39(+) and CD4(+) CD39(-)CD73(+) T cells or CD19(+) B cells isolated from blood of 30 volunteers and 14 cancer patients. CD39 and CD73 expression was evaluated by flow cytometry, Western blots, confocal microscopy or reverse transcription-polymerase chain reaction (RT-PCR). Circulating CD4(+) CD39(+) T(reg) which hydrolyzed eATP to 5'-AMP contained few intracytoplasmic granules and had low CD73 mRNA levels. Only ∼1% of these T(reg) were CD39(+) CD73(+) . In contrast, CD4(+) CD39(neg) CD73(+) T cells contained numerous CD73(+) granules in the cytoplasm and strongly expressed surface CD73. In vitro-generated T(reg) (Tr1) and most B cells were CD39(+) CD73(+) . All these CD73(+) T cell subsets and B cells hydrolyzed 5'-AMP to ADO. Exosomes isolated from plasma of normal control (NC) or cancer patients carried enzymatically active CD39 and CD73(+) and, when supplied with eATP, hydrolyzed it to ADO. Only CD4(+) CD39(+) T(reg) co-incubated with CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes produced ADO. Thus, contact with membrane-tethered CD73 was sufficient for ADO production by CD4(+) CD39(+) T(reg). In microenvironments containing CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes, CD73 is readily available to CD4(+) CD39(+) CD73(neg) T(reg) for the production of immunosuppressive ADO.

Ultrasound findings of masses of the paratesticular space.

Ultrasound is a routine investigation for the assessment of scrotal masses. Many of the detected lesions involve the paratesticular structures. The most common paratesticular masses in clinical practice are epididymal cysts and spermatoceles, but there are a large number of other pathologies that can be encountered and may result in diagnostic uncertainty. This review covers a wide range of the common and the rare, but important, causes of paratesticular masses. The ultrasound findings (both typical and atypical) of these lesions are clarified, and emphasis is given to the features that help to differentiate between them.

Caffeine protects Alzheimer's mice against cognitive impairment and reduces brain beta-amyloid production.

A recent epidemiological study suggested that higher caffeine intake over decades reduces the risk of Alzheimer's disease (AD). The present study sought to determine any long-term protective effects of dietary caffeine intake in a controlled longitudinal study involving AD transgenic mice. Caffeine (an adenosine receptor antagonist) was added to the drinking water of amyloid precursor protein, Swedish mutation (APPsw) transgenic (Tg) mice between 4 and 9 months of age, with behavioral testing done during the final 6 weeks of treatment. The average daily intake of caffeine per mouse (1.5 mg) was the human equivalent of 500 mg caffeine, the amount typically found in five cups of coffee per day. Across multiple cognitive tasks of spatial learning/reference memory, working memory, and recognition/identification, Tg mice given caffeine performed significantly better than Tg control mice and similar to non-transgenic controls. In both behaviorally-tested and aged Tg mice, long-term caffeine administration resulted in lower hippocampal beta-amyloid (Abeta) levels. Expression of both Presenilin 1 (PS1) and beta-secretase (BACE) was reduced in caffeine-treated Tg mice, indicating decreased Abeta production as a likely mechanism of caffeine's cognitive protection. The ability of caffeine to reduce Abeta production was confirmed in SweAPP N2a neuronal cultures, wherein concentration-dependent decreases in both Abeta1-40 and Abeta1-42 were observed. Although adenosine A(1) or A(2A) receptor densities in cortex or hippocampus were not affected by caffeine treatment, brain adenosine levels in Tg mice were restored back to normal by dietary caffeine and could be involved in the cognitive protection provided by caffeine. Our data demonstrate that moderate daily intake of caffeine may delay or reduce the risk of AD.

2-Hydroxyestradiol attenuates the development of obesity, the metabolic syndrome, and vascular and renal dysfunction in obese ZSF1 rats.

A pandemic of obesity is contributing importantly to the prevalence of the metabolic syndrome characterized by hypertension, insulin resistance, and hyperlipidemia. In turn, the metabolic syndrome is contributing to vascular disease and the accelerating epidemic of chronic renal failure. Currently, pharmacological approaches to attenuate obesity and its cardiovascular/renal sequelae are limited. The purpose of this study was to determine the effects of 2-hydroxyestradiol, a metabolite of 17beta-estradiol with minimal estrogenic activity, on the development of obesity, the metabolic syndrome, and heart, vascular, and renal dysfunction in obese ZSF1 rats, a well-characterized genetic model of obesity and the metabolic syndrome with concomitant heart, vascular, and kidney disease. ZSF1 rats were treated, beginning at 12 weeks of age, for 26 weeks with vehicle or 2-hydroxyestradiol (10 microg/kg/h). At baseline and after 24 weeks of treatment, animals were placed in metabolic cages, and food intake, water intake, urine output, and urinary excretion of proteins and glucose were determined. Next, in fasting animals, plasma cholesterol was measured, an oral glucose tolerance test was conducted, and total glycated hemoglobin levels were determined. At the end of the study, animals were anesthetized and instrumented for assessment of heart performance, renal hemodynamics, and mesenteric vascular reactivity. 2-Hydroxyestradiol attenuated the development of obesity and improved endothelial function, decreased nephropathy, decreased the severity of diabetes, lowered arterial blood pressure, and reduced plasma cholesterol. 2-Hydroxyestradiol may be an important lead for the development of safe and effect drugs to attenuate obesity and its metabolic, vascular, and renal sequelae.

A(1) receptor blockade induces natriuresis with a favorable renal hemodynamic profile in SHHF/Mcc-fa(cp) rats chronically treated with salt and furosemide.

Our goal was to test the hypothesis that A(1) receptor blockade induces diuresis/natriuresis with a favorable renal hemodynamic/cardiac profile in aged, lean SHHF/Mcc-fa(cp) rats, a rodent model of hypertensive dilated cardiomyopathy. Thirteen-month-old SHHF/Mcc-fa(cp) rats were pretreated for 72 h before experiments with furosemide (100 mg/kg by gavage 72, 48, and 24 h before experiments) to mimic the clinical setting of chronic diuretic therapy and were given 1% NaCl as drinking water to reduce dehydration/sodium depletion. Animals were instrumented for measurement of systemic and renal hemodynamics, renal excretory function, and cardiac performance, and baseline values were obtained during a 30-min clearance period. Animals then received either vehicle (n = 9), BG9719 [the S-enantiomer of 1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)] xanthine (also called CVT-124)] (highly selective A(1) receptor antagonist; 0.1 mg/kg bolus + 10 microg/kg/min; n = 9) or furosemide (loop diuretic; 30 mg/kg; n = 8) and measurements were repeated during four subsequent clearance periods. Both BG9719 and furosemide increased urine volume and absolute and fractional sodium excretion. BG9719 increased renal blood flow and glomerular filtration rate, but did not affect fractional potassium excretion. Furosemide decreased renal blood flow and glomerular filtration rate and increased fractional potassium excretion. Neither drug altered afterload; however, furosemide, but not BG9719, decreased preload (central venous pressure and ventricular end diastolic pressure). Neither drug altered systolic function (+dP/dt(max)); however, furosemide, but not BG9719, attenuated diastolic function (decreased -dP/dt(max), increased tau). In the setting of left ventricular dysfunction, chronic salt loading and prior loop diuretic treatment, selective A(1) receptor antagonists are effective diuretic/natriuretic agents with a favorable renal hemodynamic/cardiac performance profile.

Catecholamines abrogate antimitogenic effects of 2-hydroxyestradiol on human aortic vascular smooth muscle cells.

Catechol-O-methyltransferase (COMT)-mediated methylation of 2-hydroxyestradiol (endogenous estradiol metabolite) to 2-methoxyestradiol (angiogenesis inhibitor) may be responsible for the antimitogenic effects of 2-hydroxyestradiol on vascular smooth muscle cells (VSMCs). Catecholamines are also substrates for COMT, and increased levels of catecholamines are associated with vasoocclusive disorders. We hypothesize that catecholamines may abrogate the vasoprotective effects of 2-hydroxyestradiol by competing for COMT and inhibiting 2-methoxyestradiol formation. To test this hypothesis, we investigated the antimitogenic effects of 0.001 to 0.1 micromol/L of 2-hydroxyestradiol on human aortic VSMC proliferation (cell number and DNA synthesis), collagen synthesis, and migration in the presence and absence of catecholamines. Norepinephrine, epinephrine, and isoproterenol concentration-dependently abrogated the inhibitory effects of 2-hydroxyestradiol on cell number, DNA synthesis, collagen synthesis, and cell migration. These modulatory/attenuating effects of catecholamines were not abrogated in the presence of the alpha- and beta-adrenergic receptor antagonists, phentolamine mesylate and propranolol, respectively. In contrast to 2-hydroxyestradiol, the antimitogenic effects of 2-methoxyestradiol (0.1 micromol/L) were not attenuated by isoproterenol (1 micromol/L) or quercetin (competitive inhibitor of COMT, 10 micromol/L). Norepinephrine, epinephrine, and isoproterenol concentration-dependently (10 to 500 micromol/L) inhibited the metabolism of 2-hydroxyestradiol (0.25 to 2 micromol/L) to 2-methoxyestradiol, and the potency of the catecholamines to reverse 2-hydroxyestradiol-induced inhibition of VSMC proliferation, collagen synthesis, and migration was correlated with their ability to inhibit 2-methoxyestradiol formation. Our findings suggest that catecholamines within the vasculature may abrogate the anti-vaso-occlusive effects of estradiol and 2-hydroxyestradiol by blocking 2-methoxyestradiol formation.

Fructose-1,6-bisphosphate and MK-801 by aortic arch flush for cerebral preservation during exsanguination cardiac arrest of 20 min in dogs. An exploratory study.

In our exsanguination cardiac arrest (CA) outcome model in dogs we are systematically exploring suspended animation (SA), i.e. preservation of brain and heart immediately after the onset of CA to enable transport and resuscitative surgery during CA, followed by delayed resuscitation. We have shown in dogs that inducing moderate cerebral hypothermia with an aortic arch flush of 500 ml normal saline solution at 4 degrees C, at start of CA 20 min no-flow, leads to normal functional outcome. We hypothesized that, using the same model, but with the saline flush at 24 degrees C inducing minimal cerebral hypothermia (which would be more readily available in the field), adding either fructose-1,6-bisphosphate (FBP, a more efficient energy substrate) or MK-801 (an N-methyl-D-aspartate (NMDA) receptor blocker) would also achieve normal functional outcome. Dogs (range 19-30 kg) were exsanguinated over 5 min to CA of 20 min no-flow, and resuscitated by closed-chest cardiopulmonary bypass (CPB). They received assisted circulation to 2 h, mild systemic hypothermia (34 degrees C) post-CA to 12 h, controlled ventilation to 20 h, and intensive care to 72 h. At CA 2 min, the dogs received an aortic arch flush of 500 ml saline at 24 degrees C by a balloon-tipped catheter, inserted through the femoral artery (control group, n=6). In the FBP group (n=5), FBP (total 1440 or 4090 mg/kg) was given by flush and with reperfusion. In the MK-801 group (n=5), MK-801 (2, 4, or 8 mg/kg) was given by flush and with reperfusion. Outcome was assessed in terms of overall performance categories (OPC 1, normal; 2, moderate disability; 3, severe disability; 4, coma; 5, brain death or death), neurologic deficit scores (NDS 0-10%, normal; 100%, brain death), and brain histologic damage scores (HDS, total HDS 0, no damage; >100, extensive damage; 1064, maximal damage). In the control group, one dog achieved OPC 2, one OPC 3, and four OPC 4; in the FBP group, two dogs achieved OPC 3, and three OPC 4; in the MK-801 group, two dogs achieved OPC 3, and three OPC 4 (P=1.0). Median NDS were 62% (range 8-67) in the control group; 55% (range 34-66) in the FBP group; and 50% (range 26-59) in the MK-801 group (P=0.2). Median total HDS were 130 (range 56-140) in the control group; 96 (range 64-104) in the FBP group; and 80 (range 34-122) in the MK-801 group (P=0.2). There was no difference in regional HDS between groups. We conclude that neither FBP nor MK-801 by aortic arch flush at the start of CA, plus an additional i.v. infusion of the same drug during reperfusion, can provide cerebral preservation during CA 20 min no-flow. Other drugs and drug-combinations should be tested with this model in search for a breakthrough effect.

Cardiovascular protective effects of 17beta-estradiol metabolites.

17beta-estradiol (estradiol), the most abundant endogenous estrogen, affords cardiovascular protection. However, in a given cohort of postmenopausal women, estradiol replacement therapy provides cardiovascular protection in only a subset. The reasons for this variable action can only be understood once the mechanisms by which estradiol induces its cardiovascular protective effects are known. Because most biological effects of estradiol are mediated via estrogen receptors (ERs) and the heart and blood vessels contain both ER-alpha and ER-beta, the prevailing view is that ERs mediate estradiol-induced cardiovascular protection. However, recent findings that estradiol protects against vascular injury in arteries of mice lacking either ER-alpha or ER-beta seriously challenges this concept. Thus other non-ER mechanisms may be operative. Endogenous estradiol is enzymatically converted to several nonestrogenic metabolites, and some of these metabolites induce potent biological effects via ER-independent mechanisms. Therefore, it is conceivable that the cardiovascular protective effects of estradiol are mediated via its endogenous metabolites. On the basis of the evidence cited in this review, the cardiovascular protective effects of estradiol are both ER dependent and independent. The purpose of this article is to review the evidence regarding the cardiovascular protective effects of estradiol metabolites and to discuss the cellular, biochemical, and molecular mechanisms involved.

Intraperitoneal, but not enteric, adenosine administration improves survival after volume-controlled hemorrhagic shock in rats.

OBJECTIVE: To circumvent the potential adverse systemic side effects of adenosine, this study explored the potential benefit of intraperitoneal or enteric adenosine on survival and inflammatory responses after volume-controlled hemorrhagic shock. DESIGN: Prospective, randomized, and blinded. A three-phase, volume-controlled hemorrhagic shock model was used: hemorrhagic shock phase (120 mins), resuscitation phase (60 mins), and observation phase (72 hrs). Three groups were compared: controls, intraperitoneal adenosine, and enteric adenosine. SETTING: Animal research facility. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Starting at 20 mins of hemorrhagic shock and continuing through the resuscitation phase, all three groups received both intraperitoneal lavage and repeated bolus injections into the ileum of vehicle (normal saline) or adenosine. In the intraperitoneal adenosine group (n = 10), adenosine solution (0.1 mM) was used for intraperitoneal lavage. In the enteric adenosine group (n = 10), adenosine (1.0 mM) was injected into the ileum. Blood cytokine concentrations and leukocyte infiltration in lungs and liver were studied in 12 separate rats (control and intraperitoneal adenosine, n = 6 each) with the same hemorrhagic shock model at resuscitation time 1 hr or 4 hrs. MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure and heart rate were similar between the three groups during hemorrhagic shock and resuscitation. Potassium, lactate, and blood urea nitrogen concentrations were lower and arterial pH was higher in the intraperitoneal and enteric adenosine groups compared with the control group (both p <.05). Survival time to 72 hrs was longer in the intraperitoneal adenosine group than in the control group(p <.05). Neither plasma interleukin-1beta, interleukin-6, interleukin-10, and tumor necrosis factor-alpha concentrations nor leukocyte infiltration in the lungs and liver was different between the control and intraperitoneal adenosine groups. CONCLUSIONS: The administration of adenosine via the intraperitoneal route improves survival time after severe volume-controlled hemorrhagic shock in rats without worsening hypotension or bradycardia. This beneficial effect may not be attributable to effects of adenosine on the inflammatory response.

Role of the extracellular cAMP-adenosine pathway in renal physiology.

Adenosine exerts physiologically significant receptor-mediated effects on renal function. For example, adenosine participates in the regulation of preglomerular and postglomerular vascular resistances, glomerular filtration rate, renin release, epithelial transport, intrarenal inflammation, and growth of mesangial and vascular smooth muscle cells. It is important, therefore, to understand the mechanisms that generate extracellular adenosine within the kidney. In addition to three "classic" pathways of adenosine biosynthesis, contemporary studies are revealing a novel mechanism for renal adenosine production termed the "extracellular cAMP-adenosine pathway." The extracellular cAMP-adenosine pathway is defined as the egress of cAMP from cells during activation of adenylyl cyclase, followed by the extracellular conversion of cAMP to adenosine by the serial actions of ecto-phosphodiesterase and ecto-5'-nucleotidase. This mechanism of extracellular adenosine production may provide hormonal control of adenosine levels in the cell-surface biophase in which adenosine receptors reside. Tight coupling of the site of adenosine production to the site of adenosine receptors would permit a low-capacity mechanism of adenosine biosynthesis to have a large impact on adenosine receptor activation. The purposes of this review are to summarize the physiological roles of adenosine in the kidney; to describe the classic pathways of renal adenosine biosynthesis; to review the evidence for the existence of the extracellular cAMP-adenosine pathway; and to describe possible physiological roles of the extracellular cAMP-adenosine pathway, with particular emphasis on the kidney.

Inhibition of adenosine deaminase attenuates endotoxin-induced release of cytokines in vivo in rats.

The purpose of this study was to investigate in vivo the effects of modulating the adenosine system on endotoxin-induced release of cytokines and changes in heart performance and neurohumoral status in early, profound endotoxemia in rats. Time/pressure variables of heart performance and blood pressure were recorded continuously, and plasma levels of tumor necrosis factor alpha (TNFalpha), interleukin 1-beta (IL-1beta), plasma renin activity (PRA), and catecholamines were determined before and 90 min after administration of endotoxin (30 mg/kg of lipopolysaccharide, i.v.). Erythro-9[2-hydroxyl-3-nonyl] adenine (EHNA; an adenosine deaminase inhibitor) had no effects on measured time-pressure variables of heart performance under baseline conditions and during endotoxemia, yet significantly attenuated endotoxin-induced release of cytokines and PRA. Pretreatment with the non-selective adenosine receptor antagonist DPSPX not only prevented the effects of EHNA but also increased the basal release of cytokines and augmented PRA. At baseline, caffeine (a non-selective adenosine receptor antagonist) increased HR, +dP/dtmax, heart rate x ventricular pressure product (HR x VPSP) and +dP/dtmax normalized by pressure (+dP/dtmax/VPSP), and these changes persisted during endotoxemia. Caffeine attenuated endotoxin-induced release of cytokines and augmented endotoxin-induced increases in plasma catecholamines and PRA. Pretreatment with propranolol abolished the effects of caffeine on heart performance and neurohumoral activation during the early phase of endotoxemia. 6N-cyclopentyladenosine (CPA; selective A1 adenosine receptor agonist) induced bradicardia and negative inotropic effects, reduced work load (i.e., decreased HR, VPSP, +dP/dtmax, +dP/dtmax/VPSP and HR x VPSP) and inhibited endotoxin-induced tachycardia and renin release. CGS 21680 (selective A2A adenosine receptor agonist) decreased blood pressure under basal condition but did not potentiate decreases in blood pressure during endotoxemia. CGS 21680 completely inhibited endotoxin-induced release of TNFalpha, augmented sympathetic activity and PRA, and increased +dP/dtmax and +dP/dtmax/VPSP in the absence and presence of endotoxin. The present study provides strong evidence that inhibition of adenosine deaminase reduces cytokine release in vivo without producing significant hemodynamic and cardiac effects during the early phase of profound endotoxemia in rats. The augmented neurohumoral activation induced by caffeine is associated with decreased cytokine release induced by endotoxin. Further studies are warranted to determine the impact of these effects on cardiac function and hemodynamics in the late phase of endotoxemia.

Assessment of the effect of 2-chloroadenosine in normal rat brain using spin-labeled MRI measurement of perfusion.

Adenosine analogs such as 2-chloroadenosine are potent cerebrovasodilators. Spin-labeled MRI was used to investigate the spatial distribution, dose-response, and timing of the effect of 2-chloroadenosine on cerebral blood flow (CBF) after intraparenchymal injection into rat brain. Sprague-Dawley rats (N = 10) were injected with 2-chloroadenosine at doses of 0.3, 6.0, or 12 nmoles, or saline vehicle (2-4 microL). CBF was serially quantified in a slice through the injection site in a circular (3.6 mm diameter) region of interest (ROI) around the injection and in ipsilateral hemispheric ROIs at approximately 90 min and approximately 180 min. Marked 3.77- and 3.93-fold increases in CBF (vs. vehicle) were seen in the circular ROI at approximately 90 min and approximately 180 min after 12-nmol injection, respectively. Similarly, 2.92- and 2.78-fold increases in hemispheric CBF were observed at approximately 90 min and approximately 180 min, respectively, after injection of 12 nmoles. Linear dose-response relationships were observed at both times after injection in both ROIs (all P < 0.01). Spin-labeling MRI assessment revealed that parenchymal injection of 2-chloroadenosine produces potent, dose-dependent, and sustained vasodilation over large areas of brain. This treatment and imaging paradigm should facilitate investigation of the effect of CBF promotion in models of traumatic and ischemic brain injury.

Endogenous cyclic AMP-adenosine pathway regulates cardiac fibroblast growth.

Our previous studies show that cardiac fibroblasts express the extracellular "cAMP-adenosine pathway," that is, the generation of adenosine from extracelluar cAMP. The goal of this study was to assess whether activation of the cAMP-adenosine pathway by stimulation of endogenous cAMP synthesis regulates cardiac fibroblast growth. Cardiac fibroblasts in 3D cultures were used as the model system. Treatment of cardiac fibroblasts with forskolin, isoproterenol, or norepinephrine increased cAMP production and extracellular levels of adenosine, and these effects were prevented by inhibition of adenylyl cyclase (2',5'-dideoxyadenosine). Treatment with forskolin, isoproterenol, or norepinephrine for 24 hours inhibited DNA synthesis ((3)H-thymidine incorporation), and this effect was enhanced by combined inhibition of adenosine deaminase (erythro-9-[2-hydroxy-3-nonyl] adenine) plus adenosine kinase (iodotubercidin). Inhibition of adenylyl cyclase or adenosine receptors (1,3-dipropyl-8-p-sulfophenylxanthine or KF17837) prevented the effects of forskolin, isoproterenol, and norepinephrine on DNA synthesis. Forskolin also inhibited protein synthesis ((3)H-leucine incorporation) and cell proliferation, and these effects were blocked by adenosine receptor antagonism. Treatment of cardiac fibroblasts with norepinephrine for >48 hours but not <48 hours increased DNA synthesis, protein synthesis, and cell number. However, blockade of adenylyl cyclase or antagonism of adenosine receptors caused norepinephrine to induce proliferation in <48 hours. Our findings indicate that the endogenous cAMP-adenosine pathway regulates cardiac fibroblast growth.

Estradiol metabolites inhibit endothelin synthesis by an estrogen receptor-independent mechanism.

Estradiol inhibits endothelin-1 synthesis, an effect that may contribute to the cardiovascular protective effects of estradiol. Recent findings that estradiol inhibits neointima formation in mice lacking estrogen receptors suggests that the cardiovascular protective effects of estradiol may be mediated by means of an estrogen receptor-independent mechanism. Because 2-hydroxyestradiol and 2-methoxyestradiol, metabolites of estradiol with little/no affinity for estrogen receptors, are more potent than estradiol in inhibiting vascular smooth muscle cell growth, we investigated whether these metabolites also inhibit endothelin-1 synthesis by means of an receptor-independent mechanism. Treatment of porcine coronary artery endothelial cells for 4 to 24 hours with 0.001 to 1 micromol/L of estradiol, 2-hydroxyestradiol, or 2-methoxyestradiol concentration-dependently inhibited basal as well as serum-induced (2.5%), TNFalpha-induced (10 ng/mL), angiotensin II-induced (100 nmol/L), and thrombin-induced (4 U/mL) endothelin-1 synthesis. Estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol also inhibited serum-induced mitogen-activated protein kinase activity. As compared with estradiol, its metabolites were more potent in inhibiting endothelin-1 secretion and mitogen activated protein kinase activity. The inhibitory effects of 2-hydroxyestradiol and 2-methoxyestradiol on endothelin-1 release and mitogen-activated protein kinase activity were not blocked by ICI182780 (50 micromol/L), an estrogen receptor antagonist. Our findings indicate that the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol potently inhibit endothelin-1 synthesis by means of an estrogen receptor-independent mechanism. This effect of estradiol metabolites may be mediated by inhibition of mitogen activated protein kinase activity and may contribute to the cardioprotective effects of estradiol.

Effects of estradiol and its metabolites on glomerular endothelial nitric oxide synthesis and mesangial cell growth.

Reduced nitric oxide synthesis by glomerular endothelial cells and increased proliferation of glomerular mesangial cells is associated with glomerular remodeling that leads to accelerated glomerulosclerosis. Estradiol induces nitric oxide synthesis and slows the progression of renal disease. Because the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol are more potent than estradiol in inhibiting growth of vascular smooth muscle cells, which are phenotypically similar to mesangial cells, we compared the effects of estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol on growth of glomerular mesangial cells and on basal nitric oxide synthesis by glomerular endothelial cells. In human glomerular mesangial cells, estradiol and its metabolites concentration-dependently (1 nmol/L to 10 micromol/L) inhibited serum (2.5%)-induced DNA synthesis, cell proliferation, and collagen synthesis with the order of potency being 2-methoxyestradiol > 2-hydroxyestradiol > estradiol. ICI182780 (100 micromol/L, an estrogen receptor antagonist) blocked the growth inhibitory effects of estradiol but not 2-hydroxyestradiol or 2-methoxyestradiol. Treatment with estradiol, but not 2-hydroxyestradiol and 2-methoxyestradiol, induced nitric oxide synthesis (P<0.05, assayed by the formation of (3)H-L-citrulline from (3)H-L-arginine) in human glomerular endothelial cells, and these effects were blocked by ICI182780 and L-NMA (a nitric oxide synthesis inhibitor). In conclusion, estradiol may attenuate glomerulosclerosis by inducing nitric oxide synthesis via an estrogen receptor-dependent mechanism and by conversion to 2-hydroxyestradiol and 2-methoxyestradiol, which inhibit glomerular mesangial cell proliferation independent of estrogen receptors.

Increased 2-methoxyestradiol production in human coronary versus aortic vascular cells.

Estradiol may be cardioprotective; however, the mechanisms involved remain unclear. Recent findings that estradiol attenuates neointima formation in estrogen receptor knockout mice suggest that the cardioprotective effects of estradiol may be mediated through estrogen receptor-independent mechanisms. Because 2-methoxyestradiol, an endogenous metabolite of estradiol with no affinity for estrogen receptors, is more potent than estradiol in inhibiting vascular smooth muscle cell growth, it is feasible that 2-methoxyestradiol mediates in part the cardioprotective effects of estradiol. To address this hypothesis, we examined the kinetics of 2-methoxyestradiol synthesis in vascular smooth muscle cells and endothelial cells. In human aortic smooth muscle cells, the V(max), K(m), and V(max)/K(m) ratio values for conversion of 2-hydroxyestradiol to 2-methoxyestradiol were 19+/-0.69 pmol. min(-1) per 10(6) cells, 0.52+/-0.085 micromol/L, and 44+/-4.9 pmol. min(-1). micromol/L per 10(6) cells, respectively. In human coronary artery vascular smooth muscle cells, the V(max), K(m), and V(max)/K(m) ratio values for conversion of 2-hydroxyestradiol to 2-methoxyestradiol were 16+/-0.59 pmol. min(-1) per 10(6) cells, 0.23+/-0.011 micromol/L, and 69+/-3.6 pmol. min(-1). micromol/L per 10(6) cells, respectively (all values significantly different compared with human aortic smooth muscle cells). Also, in human aortic versus coronary artery endothelial cells, the V(max) (33+/-0.24 versus 22+/-0.33 pmol. min(-1) per 10(6) cells, respectively), K(m) (0.20+/-0.010 versus 0.099+/-0.014 micromol/L, respectively), and V(max)/K:(m) (163+/-7.7 versus 243+/-41 pmol. min(-1). micromol/L per 10(6) cells, respectively) values were significantly different. Our results indicate that vascular smooth muscle and endothelial cells effectively metabolize 2-hydroxyestradiol to 2-methoxyestradiol. The lower K(m) and higher V(max)/K(m) ratio of human coronary versus aortic cells indicate a faster rate of local metabolism of 2-hydroxyestradiol to 2-methoxyestradiol in the coronary circulation at low levels of 2-hydroxyestradiol.