Novel Mechanisms of Ozone-Induced Pulmonary Inflammation and Resolution, and the Potential Protective Role of Scavenger Receptor BI.

INTRODUCTION: Increases in ambient levels of ozone (O3), a criteria air pollutant, have been associated with increased susceptibility and exacerbations of chronic pulmonary diseases through lung injury and inflammation. O3 induces pulmonary inflammation, in part by generating damage-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and scavenger receptors (SRs). This inflammatory response is mediated in part by alveolar macrophages (AMs), which highly express PRRs, including scavenger receptor BI (SR-BI). Once pulmonary inflammation has been induced, an active process of resolution occurs in order to prevent secondary necrosis and to restore tissue homeostasis. The processes known to promote the resolution of inflammation include the clearance by macrophages of apoptotic cells, known as efferocytosis, and the production of specialized pro-resolving mediators (SPMs). Impaired efferocytosis and production of SPMs have been associated with the pathogenesis of chronic lung diseases; however, these impairments have yet to be linked with exposure to air pollutants. SPECIFIC AIMS: The primary goals of this study were: Aim 1 - to define the role of SR-BI in O3-derived pulmonary inflammation and resolution of injury; and Aim 2 - to determine if O3 exposure alters pulmonary production of SPMs and processes known to promote the resolution of pulmonary inflammation and injury. METHODS: To address Aim 1, female wild-type (WT) and SR-BI-deficient, or knock-out (SR-BI KO), mice were exposed to either O3 or filtered air. In one set of experiments mice were instilled with an oxidized phospholipid (oxPL). Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for the analyses of inflammatory and injury markers and oxPL. To estimate efferocytosis, mice were administered apoptotic cells (derived from the Jurkat T cell line) after O3 or filtered air exposure.To address Aim 2, male WT mice were exposed to either O3 or filtered air, and levels of SPMs were assessed in the lung, as well as markers of inflammation and injury in BALF. In some experiments SPMs were administered before exposure to O3or filtered air, to determine whether SPMs could mitigate inflammatory or resolution responses. Efferocytosis was measured as in Aim 1. RESULTS: For Aim 1, SR-BI protein levels increased in the lung tissue of mice exposed to O3, compared with mice exposed to filtered air. Compared with WT controls, SR-BI KO mice had a significant increase in the number of neutrophils in their airspace 24 hours post O3 exposure. The oxPL levels increased in the airspace of both WT and SR-BI KO mice after O3 exposure, compared with filtered air controls. Four hours after instillation of an oxPL, SR-BI KO mice had an increase in BALF neutrophils and total protein, and a nonsignificant increase in macrophages compared with WT controls. O3 exposure decreased efferocytosis in both WT and SR-BI KO female mice.For Aim 2, mice given SPM supplementation before O3 exposure showed significantly increased AM efferocytosis when compared with the O3exposure control mice and also showed some mitigation of the effects of O3 on inflammation and injury. Several SPMs and their precursors were measured in lung tissue using reverse-phase high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). At 24 hours after O3 exposure 14R-hydroxydocosahexaenoic acid (HDHA) and 10,17-dihydroxydocosahexaenoic acid (diHDoHE) were significantly decreased in lung tissue, but at 6 hours after exposure, levels of these SPMs increased. CONCLUSIONS: Our findings identify novel mechanisms by which O3 may induce pulmonary inflammation and also increase susceptibility to and exacerbations of chronic lung diseases.

Body piercing: more than skin deep.

Young adult populations (18-25 years of age) throughout the world have latched onto the mainstream trend of body piercing. Best health care practices for these individuals involves the knowledge of proper procedural techniques, postsite care, common complications, and treatment modalities.

Restoration of endothelium-dependent relaxation by dietary treatment of atherosclerosis.

Atherosclerosis results in impaired relaxation to acetylcholine, thrombin, and the calcium ionophore A23187, all agents that require the presence of endothelium. We now report that dietary treatment of atherosclerosis in monkeys not only produces morphological improvement of the atherosclerotic lesion but restores endothelium-dependent vascular relaxation to normal. Because the intima remains thickened after regression of atherosclerosis, these studies suggest that intimal thickening which is present in both atherosclerotic vessels and after regression of atherosclerosis does not prevent the endothelium-derived relaxing factor from reaching the underlying vascular smooth muscle.

Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of different fatty acid composition.

Six normal men were fed formula diets containing either highly saturated fat (cocoa butter, iodine value 32) or polyunsaturated fat (corn oil, iodine value 125). The sterol balance technique was used to compare the changes in serum cholesterol concentration with the excretion of fecal steroids. The method used for the analysis of fecal steroids was chemical, with a final identification and quantification by gas-liquid chromatography. It was confirmed that the chemical method for fecal steroid analysis was accurate and reproducible. The three dietary periods were each 3 wk in length. In sequence, cocoa butter (period I), corn oil, and cocoa butter (period III) were fed at 40% of the total calories. All diets were cholesterol free, contained similar amounts of plant sterols, and were identical in other nutrients. Corn oil had a hypocholesterolemic effect. Mean serum cholesterol concentrations were 222 mg/100 ml (cocoa butter, period I), 177 during corn oil, and 225 after the return to cocoa butter. Individual fecal steroids were determined from stools pooled for 7 days. Both neutral steroids and bile acids were altered significantly by dietary polyunsaturated fat. The change in bile acid excretion was considerably greater than the change in neutral steroids. Corn oil caused a greater fecal excretion of both deoxycholic and lithocholic acids. The total mean excretion (milligrams per day) of fecal steroids was 709 for cocoa butter (period I), 915 for corn oil, and 629 for the second cocoa butter period. The enhanced total fecal steroid excretion by the polyunsaturated fat of corn oil created a negative cholesterol balance vis-à-vis the saturated fat of cocoa butter. The hypocholesterolemic effect of polyunsaturated fat was associated with total fecal sterol excretion twice greater than the amount of cholesterol calculated to leave the plasma. This finding suggested possible loss of cholesterol from the tissues as well.

Hemodynamic sequelae of regression of experimental atherosclerosis.

Regression of experimental atherosclerosis is characterized by decreased intimal thickness and luminal enlargement, but intimal fibrosis becomes more dense. We tested the hypothesis that fibrosis of arteries during regression might limit vasodilator capacity and restrict hemodynamic improvement despite luminal improvement. We studied limb, coronary, and cerebral hemodynamics in 11 normal cynomolgus monkeys, 10 monkeys given an atherogenic diet for 20 mo and 8 monkeys given a regression diet for an additional 18 mo. The atherogenic diet induced lesions of moderate severity (50-60% stenosis); owing to characteristic vessel growth during the atherogenic period, luminal size did not decrease correspondingly. Regression monkeys showed typical changes of regression with luminal enlargement but increased fibrosis. The iliac artery was perfused at constant blood flow and maximal vasodilatation was produced with papaverine. Blood flow was measured with microspheres during maximal vasodilatation in the coronary bed (adenosine) and cerebral bed (hypercapnia). In normal monkeys, minimal vascular resistances were 1.95 +/- 0.19 mm Hg/ml/min X 100 g (mean +/- SE) (limb), 0.13 +/- 0.01 (coronary), and 0.44 +/- 0.02 (cerebral). In atherosclerotic monkeys minimal resistance increased (P less than 0.05) 108, 62, and 166% in the limb, coronary, and cerebral beds, respectively. In regression monkeys, minimal resistance increased from values found in atherosclerotic animals in the limb (+22%), decreased inconsistently in the coronary bed (-19%), and decreased significantly in the cerebral bed (-44%, P less than 0.05). Thus morphologic regression was accompanied by significant hemodynamic improvement during maximal dilatation only in cerebral vessels. We conclude that increases in luminal size during regression of atherosclerotic lesions may not be associated with increases in vasodilator capacity, as intimal fibrosis may limit physiologically important hemodynamic improvement.

Regulation of blood flow to the aortic media in dogs.

Morphologic observations suggest that the inner layers of the thoracic aorta in man and dog are avascular and the outer layers have vasa vasorum. It appears that vasa vasorum are essential in the thoracic aorta because their interruption produces medial necrosis. These experiments provide the first measurements of blood flow through aortic vasa vasorum and examine physiologic regulation of that flow. During control conditions the outer two-thirds of the media of the thoracic aorta received 10 ml/min per 100 g blood flow through vasa vasorum. Flow to the inner third of the aorta was 1 ml/min per 100 g. Flow to both the inner and outer media of the abdominal aorta was less than 1 ml/min per 100 g. Adenosine increased blood flow to vasa vasorum in the outer media of the thoracic aorta from 7 to 18 ml/min per 100 g, but did not increase flow to the inner layers of the aorta. Hemorrhagic hypotension decreased flow in the outer media of the thoracic aorta from 14 to 2 ml/min per 100 g. Acute hypertension failed to increase blood flow through vasa vasorum, as conductance decreased significantly. These studies indicate that vasa vasorum provide a considerable amount of blood flow to the outer layers of the thoracic aorta. The vessels are responsive to physiologic stimuli because they dilate during infusion of adenosine and constrict during both hemorrhagic hypotension and acute hypertension. We speculate that the failure of blood flow to the aortic wall to increase during acute hypertension might, if it were sustained, contribute to aortic medial necrosis.

A defect in mobilization of cholesteryl esters in rabbit macrophages.

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholesterolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A:cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A:cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.

Increased production of lipoxygenase products by cholesterol-rich mouse macrophages.

The metabolism of arachidonic acid by cholesterol-enriched resident mouse peritoneal macrophages was investigated. The amounts of monohydroxyeicosatetraenoic acid (mono-HETE) produced by the cholesterol-rich macrophages were 2.5-fold greater when compared to control macrophages. The major lipoxygenase product, identified by high-performance liquid chromatography in both macrophages was 12-HETE. Since macrophages are important participants in the formation of atheromatous lesions, the increased metabolism of arachidonic acid to HETE products by cholesterol-rich macrophages could contribute to the initiation and progression of the atherosclerotic process.

Coronary arterial remodeling studied by high-frequency epicardial echocardiography: an early compensatory mechanism in patients with obstructive coronary atherosclerosis.

Coronary arterial remodeling is a compensatory mechanism that may limit the adverse effects of coronary obstructive lesions by expansion of the entire vascular segment. To determine if this compensatory anatomic change occurs in patients, high-frequency epicardial echocardiography using a 12 MHz transducer was performed during open heart surgery in 33 patients (10 with normal coronary arteries undergoing valvular surgery and 23 with coronary atherosclerosis). From stop-frame videotape high-frequency epicardial echocardiographic images, cross-sectional measurements of luminal area and total arterial area (lumen, intima, media and dense adventitia) were made in the patients with atherosclerosis at the site of arterial lesions and from the most proximal portion of the same artery. Remodeling was defined as enlargement of the total arterial area. In normal arteries measurements were made from proximal and midarterial locations. In the patients with normal coronary arteries, total arterial area, as determined by high-frequency echocardiography, decreased from the proximal site to the midportion of the artery (from 10.4 +/- 0.9 to 8.4 +/- 1.0 mm2, p less than 0.05); luminal area also decreased (from 6.0 +/- 0.6 to 4.5 +/- 0.7 mm2, p less than 0.05). In patients with coronary arterial lesions, luminal area also decreased from the proximal site to the arterial lesion site (from 5.3 +/- 0.6 to 2.3 +/- 0.3 mm2, p less than 0.05), but total arterial area increased (from 11.6 +/- 1.0 to 13.0 +/- 1.0 mm2, p less than 0.05). Of the 25 coronary arteries evaluated, only 4 had angiographic evidence of coronary collateral formation. These data indicate that coronary arterial remodeling is an important compensatory mechanism in obstructive coronary disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Variable morphology of coronary atherosclerosis: characterization of atherosclerotic plaque and residual arterial lumen size and shape by epicardial echocardiography.

The purpose of this study was to evaluate the in vivo characteristics of coronary atherosclerosis by using high frequency epicardial echocardiography. High frequency epicardial echocardiography was used to evaluate residual lumen and wall morphology at the sites of maximal coronary atherosclerosis in 26 patients undergoing coronary artery bypass grafting. The maximal/minimal wall thickness ratio was 3.1 +/- 0.2 (mean +/- SEM) with a large range (1.3 to 7.5). Portions of the wall were normal in 16 of 31 lesions; the percent normal circumference ranged from 9% to 85%. Maximal/minimal lumen diameter ratio was 1.5 +/- 0.1 (range 1.1 to 2.9). The shape of the residual coronary lumen was noncircular in 16 lesions: oval in 13 and complex in 3. The residual coronary lumen was eccentrically placed within six arteries. These data emphasize the variability of residual lumen and wall geometry in atherosclerosis.

Arterial uptake indices of low density lipoproteins after fatty streak formation in Cynomolgus monkeys.

To evaluate changes in arterial wall uptake of an atherogenic lipoprotein in early atherogenesis the uptake of labelled low density lipoprotein was measured in four segments of aorta in Cynomolgus monkeys with diet induced fatty streaks. The influx rate of low density lipoprotein was estimated from a simple kinetic model. The mean influx rate, expressed as plasma equivalents of tracer, was 69 nl.cm-2 per hour in fatty streak monkeys and 28 nl.cm-2 per hour in controls (p less than 0.001). Using additional assumptions, provisional estimates of the rates of apparent efflux were calculated. In fatty streak monkeys the mean apparent efflux rate, relative to the accumulation of tracer in the wall, did not differ significantly from the efflux rate in controls (8.7% per hour vs 7.9% per hour, p greater than 0.05). Thus intimal permeability to low density lipoprotein is increased at the fatty streak stage of lesion formation but apparent efflux may not be changed. The data fit the hypothesis that relative failure of efflux is a major cause of excess low density lipoprotein accumulation in the vessel wall in hypercholesterolaemia.

Response of the mesenteric circulation to serotonin in normal and atherosclerotic monkeys: implications for the pathogenesis of non-occlusive intestinal ischaemia.

We tested the hypothesis that atherosclerosis potentiates vasoconstrictor responses in the mesenteric circulation to stimulation of serotonergic and alpha-adrenergic receptors. In normal monkeys, infusion of serotonin had little effect on blood flow to the stomach, duodenum, and colon. In contrast, in atherosclerotic monkeys, serotonin produced a modest decrease in blood flow to the stomach and duodenum, and virtually abolished blood flow to the colon. Vasoconstrictor responses to phenylephrine in the stomach and duodenum were not altered by atherosclerosis, but responses were potentiated in the colon of atherosclerotic monkeys. In summary, atherosclerosis greatly potentiates vasoconstrictor responses to serotonin in the mesenteric circulation, particularly in vessels to the colon. We speculate that release of serotonin during adherence and aggregation of platelets at atherosclerotic lesions, coupled with augmented vasoconstrictor responses to serotonin, may play a role in the pathogenesis of non-occlusive mesenteric ischaemia.

Effects of chronic hypertension on vasa vasorum in the thoracic aorta.

The outer layers of the thoracic aorta receive substantial blood flow through vasa vasorum within the aortic wall. Flow delivered via these channels is functionally important because medial necrosis occurs when vasa vasorum are ligated. If flow through vasa vasorum is limited in chronic hypertension, this could contribute to medial necrosis and, perhaps, aortic dissection. In these experiments, flow and conductance in vasa vasorum were assessed in twelve awake dogs with renal hypertension (arterial pressure = 127 +/- 4 mmHg [mean +/- SE]) and nine normotensive controls (arterial pressure = 100 +/- 3 mmHg [P less than 0.001]). At rest, blood flow delivered via vasa vasorum to the thoracic aorta was similar in hypertensive and normotensive dogs (5.2 +/- 0.9 and 4.8 +/- 0.4 ml . min-1 X 100 g-1 respectively). Thus, in hypertensive dogs, conductance of the vasa vasorum decreased to maintain flow constant. During maximal dilatation induced by iv adenosine (4.7 mumol . kg-1 per min) flow delivered via vasa vasorum increased by 100% in both hypertensive and normotensive dogs. Calculations of maximum conductance indicate that vasodilator capacity was decreased by 67% in vasa vasorum of hypertensive dogs. These data suggest that vasodilator capacity of vasa vasorum in the thoracic aorta is limited in chronic hypertension. This abnormality could contribute to the pathogenesis of medial necrosis and aortic dissection in hypertensive patients.

Advancing Behavioral HIV Prevention: Adapting an Evidence-Based Intervention for People Living with HIV and Alcohol Use Disorders.

Alcohol use disorders (AUDs) are highly prevalent among people living with HIV/AIDS (PLWHA) and are associated with increased HIV risk behaviors, suboptimal treatment adherence, and greater risk for disease progression. We used the ADAPT-ITT strategy to adapt an evidence-based intervention (EBI), the Holistic Health Recovery Program (HHRP+), that focuses on secondary HIV prevention and antiretroviral therapy (ART) adherence and apply it to PLWHA with problematic drinking. Focus groups (FGs) were conducted with PLWHA who consume alcohol and with treatment providers at the largest HIV primary care clinic in New Orleans, LA. Overall themes that emerged from the FGs included the following: (1) negative mood states contribute to heavy alcohol consumption in PLWHA; (2) high levels of psychosocial stress, paired with few adaptive coping strategies, perpetuate the use of harmful alcohol consumption in PLWHA; (3) local cultural norms are related to the permissiveness and pervasiveness of drinking and contribute to heavy alcohol use; (4) healthcare providers unanimously stated that outpatient options for AUD intervention are scarce, (5) misperceptions about the relationships between alcohol and HIV are common; (6) PLWHA are interested in learning about alcohol's impact on ART and HIV disease progression. These data were used to design the adapted EBI.

Sick vessel syndrome. Recovery of atherosclerotic and hypertensive vessels.

This review describes vascular changes in atherosclerotic and hypertensive vessels as well as effects of treatment. Changes in vascular structure in both atherosclerosis and hypertension are characterized by thickening of the vessel wall and vascular "remodeling." Remodeling tends to preserve the size of the lumen in atherosclerotic vessels and results in a smaller lumen in hypertensive vessels. Changes in vascular function are characterized by preservation of smooth muscle relaxation, with the exception of activity of ATP-sensitive potassium channels, and dysfunction of endothelium. Regression of atherosclerosis, by treatment of hyperlipidemia, results in quite rapid removal of lipid from the vessel wall but with inconsistent improvement in maximal vasodilator capacity. In contrast, endothelial function improves during regression of atherosclerosis, and hyperresponsiveness to serotonin subsides rapidly. Effective treatment of hypertension produces regression of vascular hypertrophy, and some approaches (especially angiotensin-converting enzyme inhibitors) are effective in correcting vascular remodeling. Endothelium-dependent relaxation generally improves during antihypertensive treatment. Reduction in pulse pressure may be more important than reduction in mean arterial pressure in reversing the structural and functional abnormalities of hypertensive vessels.

Hypothesis: vasoconstriction contributes to amaurosis fugax.

Platelets play a critical role in the pathophysiology of amaurosis fugax. Emboli to retinal vessels apparently produce amaurosis but, in addition, we propose that augmented vasoconstrictor responses and vasospasm may contribute to amaurosis. In this study we tested the hypothesis that constrictor responses of retinal vessels to serotonin, which is released when platelets aggregate, are potentiated in experimental atherosclerosis. Blood flow to the retina was measured in normal and atherosclerotic cynomolgus monkeys. In normal monkeys, infusion of serotonin did not alter flow to the retina. In atherosclerotic monkeys, infusion of serotonin reduced retinal blood flow (in milliliters per minute per 100 g) from 66 +/- 7 (mean +/- SE) to 5 +/- 2. Infusion of serotonin in atherosclerotic monkeys abolished the retinal response to light. Thus, atherosclerosis greatly potentiates constrictor responses to serotonin in the retinal circulation and produces a profound but reversible impairment of retinal function. We propose that altered responses to vasoactive substances that are released by platelets may contribute to the pathogenesis of amaurosis fugax.

Specific metabolic effects imposed by Streptococcus pneumoniae upon the response to femoral fracture in the rat.

The possible potentiation of an infection upon the metabolic consequences of trauma was tested in rats using a 2 X 2 block design which included control, femoral fracture, pneumococcal infection, and fracture plus infection groups. Infection introduced unique metabolic effects different from those of starvation, femoral fracture, or both together. Infection-induced effects included an accelerated conversion of 14C-alanine to glucose, higher serum haptoglobin, alpha2-macrofetoprotein, copper, and ceruloplasmin values, and lower serum iron, zinc, and transferrin concentrations. The first three of these infection-induced effects were diminished in rats with a femoral fracture. No measured effect of infection was increased in traumatized rats.

Effects of sympathetic nerves on collateral vessels in the limb of atherosclerotic primates.

This study was performed to examine effects of sympathetic nerves on collateral vessels in the limb. We studied normal (N) and atherosclerotic (AS) cynomolgus monkeys that were fed atherogenic diet for 21 months. A common iliac artery was ligated 13 months before hemodynamic measurements. Using histofluorescence microscopy, a plexus of noradrenergic nerves was identified in the adventitia of collateral vessels. We measured blood flow to the limb with microspheres, and the pressure gradient from aorta to the iliac artery beyond the occlusion. The lumbar sympathetic chain was stimulated electrically at 3 Hz (SNS-3) and 15 Hz (SNS-15). In normal monkeys, conductance of collateral vessels (in ml/min per 100 g per 100 mm Hg) was 19 +/- 3.6 (mean +/- SE) during control, 14 +/- 1.6 during SNS-3, and 9.8 +/- 0.9 during SNS-15 (P less than 0.05 vs control). In AS monkeys, collateral conductance was 12 +/- 2.9 during control, 7.5 +/- 1.7 during SNS-3 and 3.9 +/- 1.8 during SNS-15 (P less than 0.05). In summary, collateral vessels in the limb are innervated and sympathetic stimulation produces pronounced constriction of collateral vessels in both normal and atherosclerotic monkeys. Thus, the effectiveness of collateral vessels in maintaining blood flow to the limb may be compromised by increased activity of sympathetic nerves.

Diffusional support of the thoracic aorta in atherosclerotic monkeys.

We have demonstrated previously that there is increased blood flow through vasa vasorum in the aorta of atherosclerotic monkeys. In this study, a new method was used to examine diffusional support to the wall of blood vessels. Our goal was to determine whether proliferation of vasa vasorum in the outer media contributes to increased diffusional support of the atherosclerotic aorta and whether there are focal or homogeneous increases in diffusion to the aortic wall. Cynomolgus monkeys were fed normal or atherogenic diet for 1.5 or 4 years. Diffusional support of the thoracic aorta was determined by measuring the concentration of iodo[14C]antipyrine in the aortic media. The findings suggest that: 1) vasa vasorum provide only minimal nutritional support to the thoracic aorta of normal monkeys and monkeys with moderately severe atherosclerosis, and 2) in monkeys with severe atherosclerosis, diffusional support to the thoracic aorta is strikingly heterogeneous, ranging from minimal to large. We speculate that minimal diffusional support to the outer layers of the thickened atherosclerotic aorta may contribute to development of focal medial necrosis.