Showing papers in "Journal of Clinical Investigation in 1991"

Role of oxidized low density lipoprotein in atherogenesis.

Abstract: Evidence to support an important role of oxidative modification in mediating the atherogenicity of LDL continues to grow. New hypotheses suggest mechanisms by which Ox-LDL or products of Ox-LDL can affect many components of the atherogenic process, including vasomotor properties and thrombosis, as well as lesion initiation and progression itself. These ideas suggest new approaches, that in combination with lowering of plasma cholesterol, could lead to the prevention of atherosclerosis and its complications.

Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide.

Abstract: Using a specific radioimmunoassay for human brain natriuretic peptide (hBNP) with a monoclonal antibody, we have investigated its synthesis, secretion, and clearance in comparison with those of atrial natriuretic peptide (ANP) in normal subjects and patients with congestive heart failure (CHF). Mean BNP-like immunoreactivity (-LI) levels in normal atrium and ventricle were 250 and 18 pmol/g, respectively. The plasma BNP-LI level in normal subjects was 0.90 +/- 0.07 fmol/ml, which was 16% of the ANP-LI level. In contrast, the plasma BNP-LI level markedly increased in patients with CHF in proportion to its severity, and surpassed the ANP-LI level in severe cases. There was a significant step-up of the plasma BNP-LI level in the coronary sinus (CS) compared with that in the aortic root (Ao) and the difference between these BNP-LI levels, delta(CS-Ao)BNP, also increased with the severity of CHF. In addition, the step-up of the BNP-LI level in the anterior interventricular vein [delta(AIV-Ao)BNP] was comparable to delta(CS-Ao)BNP, indicating that BNP is secreted mainly from the ventricle. Predominant BNP synthesis in the ventricle was also confirmed by Northern blot analysis. Catheterization and pharmacokinetic studies revealed that hBNP is cleared from the circulation more slowly than alpha-hANP; this was in part attributed to lower (about 7%) binding affinity of hBNP to clearance receptors than that of alpha-hANP. A predominant molecular form of BNP-LI in the heart and plasma was a 3-kD form corresponding to hBNP. These results indicate that BNP is a novel cardiac hormone secreted predominantly from the ventricle, and that the synthesis, secretion and clearance of BNP differ from those of ANP, suggesting discrete physiological and pathophysiological roles of BNP in a dual natriuretic peptide system.

Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes.

Abstract: Nitric oxide (an endothelium-derived relaxing factor) induces smooth muscle relaxation and is an important mediator in the regulation of vascular tone. Advanced glycosylation end products, the glucose-derived moieties that form nonenzymatically and accumulate on long-lived tissue proteins, have been implicated in many of the complications of diabetes and normal aging. We demonstrate that advanced glycosylation products quench nitric oxide activity in vitro and in vivo. Acceleration of the advanced glycosylation process in vivo results in a time-dependent impairment in endothelium-dependent relaxation. Inhibition of advanced glycosylation with aminoguanidine prevents nitric oxide quenching, and ameliorates the vasodilatory impairment. These results implicate advanced glycosylation products as important modulators of nitric oxide activity and endothelium-dependent relaxation.

The plasminogen activator/plasmin system.

Abstract: proteases and inhibitors has been shown to be under the control of hormones and growth factors. Plasminogen activators (PAs) and their inhibitors (PAIs) are thought to be key participants in the balance ofproteolytic and antiproteolytic activities that regulates matrix turnover. This article summarizes the evidence that supports this contention, discusses the role ofPAspecific cell surface binding sites, and also draws attention to a number of instances in which the presence ofPAs cannot be reconciled with an exclusive function in ECM degradation.

Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans

Abstract: Hyperinsulinemia may contribute to hypertension by increasing sympathetic activity and vascular resistance We sought to determine if insulin increases central sympathetic neural outflow and vascular resistance in humans We recorded muscle sympathetic nerve activity (MSNA; microneurography, peroneal nerve), forearm blood flow (plethysmography), heart rate, and blood pressure in 14 normotensive males during 1-h infusions of low (38 mU/m2/min) and high (76 mU/m2/min) doses of insulin while holding blood glucose constant Plasma insulin rose from 8 +/- 1 microU/ml during control, to 72 +/- 8 and 144 +/- 13 microU/ml during the low and high insulin doses, respectively, and fell to 15 +/- 6 microU/ml 1 h after insulin infusion was stopped MSNA, which averaged 215 +/- 15 bursts/min in control, increased significantly (P less than 0001) during both the low and high doses of insulin (+/- 54 and +/- 93 bursts/min, respectively) and further increased during 1-h recovery (+152 bursts/min) Plasma norepinephrine levels (119 +/- 19 pg/ml during control) rose during both low (258 +/- 25; P less than 002) and high (285 +/- 95; P less than 001) doses of insulin and recovery (316 +/- 23; P less than 001) Plasma epinephrine levels did not change during insulin infusion Despite the increased MSNA and plasma norepinephrine, there were significant (P less than 0001) increases in forearm blood flow and decreases in forearm vascular resistance during both doses of insulin Systolic pressure did not change significantly during infusion of insulin and diastolic pressure fell approximately 4-5 mmHg (P less than 001) This study suggests that acute increases in plasma insulin within the physiological range elevate sympathetic neural outflow but produce forearm vasodilation and do not elevate arterial pressure in normal humans

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Abstract: Studies of the mode of action of the bisphosphonate alendronate showed that 1 d after the injection of 0.4 mg/kg [3H]alendronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. Silver grains were seen above the osteoclasts and no other cells. 6 d later the label was 600-1,000 microns away from the epiphyseal plate and buried inside the bone, indicating normal growth and matrix deposition on top of alendronate-containing bone. Osteoclasts from adult animals, infused with parathyroid hormone-related peptide (1-34) and treated with 0.4 mg/kg alendronate subcutaneously for 2 d, all lacked ruffled border but not clear zone. In vitro alendronate bound to bone particles with a Kd of approximately 1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5 binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 x 10(-3) fmol/microns 2, which would produce a concentration of 0.1-1 mM in the resorption space if 50% were released. At these concentrations membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts.

Monocyte chemoattractant protein-1 in human atheromatous plaques.

Abstract: Monocytes appear to be central to atherogenesis both as the progenitors of foam cells and as a potential source of growth factors mediating intimal hyperplasia, but the chemical messages which stimulate the influx of monocytes into human atheroma remain unknown. Monocyte chemoattractant protein-1 (MCP-1) is a recently described molecule with powerful monocyte chemotactic activity expressed by monocytes, vascular endothelial cells, and smooth muscle cells in culture. To begin to address the role of MCP-1 in vivo, we examined 10 normal arteries and 14 diseased human arteries for MCP-1 expression by in situ hybridization. MCP-1 mRNA was detected in 16% of 10,768 cells counted in human carotid endarterectomy specimens with highest expression seen in organizing thrombi (33%) and in macrophage rich areas bordering the necrotic lipid core (24%) as compared to the fibrous cap (8%) and the necrotic lipid core itself (5%). Based on immunohistochemical staining of serial sections and on cell morphology, MCP-1 mRNA appeared to be expressed by vascular smooth muscle cells (VSMC), mesenchymal appearing intimal cells (MICs), and macrophages. By contrast, few cells expressing MCP-1 mRNA were found in normal arteries (less than 0.1%). These data suggest a potential role for MCP-1 in mediating monocytic infiltration of the artery wall.

Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma.

Abstract: We have attempted to identify mRNA for IL-5 in endobronchial mucosal biopsies from asthmatics and controls, using the technique of in situ hybridization. Bronchial biopsies were obtained from 10 asthmatics and 9 nonatopic normal controls. A radio-labeled cRNA probe was prepared from an IL-5 cDNA and hybridized to permeabilized sections. These were washed extensively before processing for autoradiography. An IL-5-producing T cell clone derived from a patient with the hyper-IgE syndrome was used as a positive control. As a negative control, sections were also treated with a "sense" IL-5 probe. Specific hybridization signals for IL-5 mRNA were demonstrated within the bronchial mucosa in 6 out of the 10 asthmatic subjects. Cells exhibiting hybridization signals were located beneath the epithelial basement membrane. In contrast, there was no hybridization in the control group. No hybridization was observed with the sense probe. The six IL-5 mRNA-positive asthmatics tended to have more severe disease than the negative asthmatics, as assessed by symptoms and lung function, and showed a significant increase in the degree of infiltration of the bronchial mucosa by secreting (EG2+) eosinophils and activated (CD25+) T lymphocytes. Within the subjects who showed positive IL-5 mRNA, there was a correlation between IL-5 mRNA expression and the number of CD25+ and EG2+ cells and total eosinophil count. This study provides evidence for the cellular localization of IL-5 mRNA in the bronchial mucosa of asthmatics and supports the concept that this cytokine regulates eosinophil function in bronchial asthma.

Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions.

Abstract: Multiple sclerosis (MS) brain tissue, spleen, and PBMC were studied using immunocytochemistry and FACS for immunoreactivity for lymphotoxin (LT) and TNF. Both cytokines were identified in acute and chronic active MS lesions but were absent from chronic silent lesions. LT was associated with CD3+ lymphocytes and Leu-M5+ microglia cells at the lesion edge and to a lesser extent, in adjacent white matter. TNF was associated with astrocytes in all areas of the lesion, and with foamy macrophages in the center of the active lesion. In acute lesions, immunoreactivity for TNF in endothelial cells was noted at the lesion edge. No LT or TNF reactivity was detected in Alzheimer's or Parkinson's disease brain tissues but was present at lower levels in central nervous system (CNS) tissue from other inflammatory conditions, except for adrenoleucodystrophy which displayed high levels of LT in microglia. No increase in LT and TNF reactivity was detected in spleen and PBMC of MS patients suggesting specific reactivity within the CNS. These results indicate that LT and TNF may be involved in the immunopathogenesis of MS, and can be detected in both inflammatory cells and cells endogenous to the CNS.

Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein

Abstract: Incubation of cocultures of human aortic endothelial (HAEC) and smooth muscle cells (HASMC) with LDL in the presence of 5-10% human serum resulted in a 7.2-fold induction of mRNA for monocyte chemotactic protein 1 (MCP-1), a 2.5-fold increase in the levels of MCP-1 protein in the coculture supernatants, and a 7.1-fold increase in the transmigration of monocytes into the subendothelial space of the cocultures. Monocyte migration was inhibited by 91% by antibody to MCP-1. Media collected from the cocultures that had been incubated with LDL induced target endothelial cells (EC) to bind monocyte but not neutrophil-like cells. Media collected from cocultures that had been incubated with LDL-induced monocyte migration into the subendothelial space of other cocultures that had not been exposed to LDL. In contrast, media from separate cultures of EC or smooth muscle cells (SMC) containing equal number of EC or SMC compared to coculture and incubated with the same LDL did not induce monocyte migration when incubated with the target cocultures. High density lipoprotein HDL, when presented to cocultures together with LDL, reduced the increased monocyte transmigration by 91%. Virtually all of the HDL-mediated inhibition was accounted for by the HDL2 subfraction. HDL3 was essentially without effect. Apolipoprotein AI was also ineffective in preventing monocyte transmigration while phosphatidylcholine liposomes were as effective as HDL2 suggesting that lipid components of HDL2 may have been responsible for its action. Preincubating LDL with beta-carotene or with alpha-tocopherol did not reduce monocyte migration. However, pretreatment of LDL with probucol or pretreatment of the cocultures with probucol, beta-carotene, or alpha-tocopherol before the addition of LDL prevented the LDL-induced monocyte transmigration. Addition of HDL or probucol to LDL after the exposure to cocultures did not prevent the modified LDL from inducing monocyte transmigration in fresh cocultures. We conclude that cocultures of human aortic cells can modify LDL even in the presence of serum, resulting in the induction of MCP-1, and that HDL and antioxidants prevent the LDL induced monocyte transmigration.

Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production.

Abstract: A large series of T cell clones (TCC) specific for purified protein derivative (PPD) of Mycobacterium tuberculosis (total 60) or Toxocara canis excretory/secretory (TES) antigen (total 69) were established from the peripheral blood of two healthy individuals and analyzed for their profile of cytokine production in response to stimulation with either the specific antigen or the polyclonal activator phorbol myristate acetate plus anti-CD3 antibody. Under both these experimental conditions, the great majority of PPD-specific TCC secreted IL-2 and IFN-gamma but not, or limited amounts of, IL-4 and IL-5. In contrast, most TES-specific TCC secreted IL-4 and IL-5 but not, or limited amounts of, IL-2 and IFN-gamma. PPD-specific TCC that failed to secrete IL-4 and IL-5, and TES-specific TCC that failed to secrete IL-2 and IFN-gamma, were found to lack transcripts for IL-4 and IL-5, or for IL-2 and IFN-gamma, respectively. During the course of the study, over a 6-mo period, the functional phenotype of both TES- and PPD-specific TCC was repeatedly assessed and remained constant. These data demonstrate that T cells with stable Th1 or Th2 functional pattern exist not only in mice but also in humans and suggest that in the course of natural immunization certain infectious agents preferentially expand T cell subsets with stable and definite profile of cytokine production.

Dysregulation of in vitro cytokine production by monocytes during sepsis.

Abstract: The production by monocytes of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) in intensive care unit (ICU) patients with sepsis syndrome (n = 23) or noninfectious shock (n = 6) is reported. Plasma cytokines, cell-associated cytokines within freshly isolated monocytes and LPS-induced in vitro cytokine production were assessed at admission and at regular intervals during ICU stay. TNF alpha and IL-6 were the most frequently detected circulating cytokines. Despite the fact that IL-1 alpha is the main cytokine found within monocytes upon in vitro activation of cells from healthy individuals, it was very rarely detected within freshly isolated monocytes from septic patients, and levels of cell-associated IL-1 beta were lower than those of TNF alpha. Cell-associated IL-1 beta and TNF alpha were not correlated with corresponding levels in plasma. Upon LPS stimulation, we observed a profound decrease of in vitro IL-1 alpha production by monocytes in all patients, and of IL-1 beta, IL-6, and TNF alpha in septic patients. This reduced LPS-induced production of cytokines was most pronounced in patients with gram-negative infections. Finally, monocytes from survival patients, but not from nonsurvival ones recovered their capacity to produce normal amounts of cytokines upon LPS stimulation. In conclusion, our data indicate an in vivo activation of circulating monocytes during sepsis as well as in noninfectious shock and suggest that complex regulatory mechanisms can downregulate the production of cytokines by monocytes during severe infections.

Killing of gram-negative bacteria by lactoferrin and lysozyme.

Abstract: Although lactoferrin has antimicrobial activity, its mechanism of action is not full defined. Recently we have shown that the protein alters the Gram-negative outer membrane. As this membrane protects Gram-negative cells from lysozyme, we have studied whether lactoferrin's membrane effect could enhance the antibacterial activity of lysozyme. We have found that while each protein alone is bacteriostatic, together they can be bactericidal for strains of V. cholerae, S. typhimurium, and E. coli. The bactericidal effect is dose dependent, blocked by iron saturation of lactoferrin, and inhibited by high calcium levels, although lactoferrin does not chelate calcium. Using differing media, the effect of lactoferrin and lysozyme can be partially or completely inhibited; the degree of inhibition correlating with media osmolarity. Transmission electron microscopy shows that E. coli cells exposed to lactoferrin and lysozyme at 40 mOsm become enlarged and hypodense, suggesting killing through osmotic damage. Dialysis chamber studies indicate that bacterial killing requires direct contact with lactoferrin, and work with purified LPS suggests that this relates to direct LPS-binding by the protein. As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense.

Hypoxia induces endothelin gene expression and secretion in cultured human endothelium.

Abstract: Hypoxia in vivo is associated with constriction of the distal vasculature in the lung. Uniquely situated at the interface between blood and the vessel wall proper, the vascular endothelium may release vasoactive mediators in the setting of hypoxia. Endothelin-1 is a potent vasoconstrictor released by endothelial cells that could function as a paracrine regulator of vascular tone. We found that physiologic low oxygen tension (PO2 = 30 Torr) increased endothelin secretion from cultured human endothelial cells four to eightfold above the secretion rate at ambient oxygen tension. This increase in secretion was accompanied by a corresponding increase in the transcriptional rate of the preproendothelin gene resulting in increased steady-state mRNA levels of preproendothelin. In contrast, the transcription of a number of other growth-factor-encoding genes, including transforming growth factor-beta, was unaffected by hypoxia. Endothelin transcript production increased within 1 h of hypoxia and persisted for at least 48 h. In addition, the stimulatory effects of low oxygen tension on endothelin mRNA levels were reversible upon reexposure to 21% oxygen environments. These findings suggest a role for endothelin in the control of regional blood flow in the vasculature in response to changes in oxygen tension.

Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome.

Abstract: Acute Respiratory Distress Syndrome (ARDS) is characterized by lung injury and damage to the alveolar type II cells. This study sought to determine if endogenous surfactant is altered in ARDS. Bronchoalveolar lavage was performed in patients at-risk to develop ARDS (AR, n = 20), with ARDS (A, n = 66) and in normal subjects (N, n = 29). The crude surfactant pellet was analyzed for total phospholipids (PL), individual phospholipids, SP-A, SP-B, and minimum surface tension (STmin). PL was decreased in both AR and A (3.48 +/- 0.61 and 2.47 +/- 0.40 mumol/ml, respectively) compared to N (7.99 +/- 0.60 mumol/ml). Phosphatidylcholine was decreased in A (62.64 +/- 2.20% PL) compared to N (76.27 +/- 2.05% PL). Phosphatidylglycerol was 11.58 +/- 1.21% PL in N and was decreased to 6.48 +/- 1.43% PL in A. SP-A was 123.64 +/- 20.66 micrograms/ml in N and was decreased to 49.28 +/- 21.68 micrograms/ml in AR and to 29.88 +/- 8.49 micrograms/ml in A. SP-B was 1.28 +/- 0.33 micrograms/ml in N and was decreased to 0.57 +/- 0.24 micrograms/ml in A. STmin was increased in AR (15.1 +/- 2.53 dyn/cm) and A (29.04 +/- 2.05 dyn/cm) compared to N (7.44 +/- 1.61 dyn/cm). These data demonstrate that the chemical composition and functional activity of surfactant is altered in ARDS. Several of these alterations also occur in AR, suggesting that these abnormalities occur early in the disease process.

The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress.

Abstract: Exposure of platelets to shear stress leads to aggregation in the absence of exogenous agonists. We have now found that different adhesive proteins and platelet membrane glycoproteins are involved in aggregation depending on the shear stress conditions and the concentration of divalent cations in the medium. When blood is collected with trisodium citrate as anticoagulant, which causes a decrease in the levels of external ionized calcium ([Ca2+]o), platelet aggregation can be induced under low shear force (12 dyn/cm2) and is mediated by fibrinogen binding to the glycoprotein IIb-IIIa complex. Aggregates formed under these conditions are not stable, and when shear force is increased to 68 dyn/cm2, disaggregation results. By contrast, platelets from blood collected with hirudin as anticoagulant, wherein [Ca2+]o is within normal plasma levels, do not undergo low shear-induced aggregation; however, after exposure to a shear force above 80 dyn/cm2, aggregation is observed but only when von Willebrand factor is present and can interact with both its platelet binding sites, glycoprotein Ib-IX and glycoprotein IIb-IIIa. Fibrinogen is not involved in high shear-induced aggregation which, in fact, occurs normally in patients with severe afibrinogenemia. Thus, von Willebrand factor in the absence of exogenous agonists can mediate platelet aggregation in experimental conditions that may mimic the hemorheological situation of partially occluded arteries. This pathway of platelet aggregation involving only one adhesive ligand and two membrane adhesion receptors may play a relevant role in thrombogenesis.

Interleukin 1 receptor antagonist. A new member of the interleukin 1 family.

Abstract: This review has summarized recent information derived from many laboratories on the discovery, characteristics, and properties of a new member of the IL-1 family, IL-1 receptor antagonist. In addition to information, an emphasis has been placed on unanswered questions and new concepts. The existence of this first-described naturally occurring specific cytokine receptor antagonist may lead to a different perspective on the cytokine network. A major unanswered question emphasized throughout this review, that now can be addressed more directly, concerns what are the physiological roles of members of the IL-1 family. Although IL-1 beta is presumed to function primarily as an extracellular cytokine, this molecule lacks a leader peptide, is synthesized and handled by the cells in a manner suggestive of a cytoplasmic (not secretory) protein, and may only be released after cellular injury. Furthermore, although IL-1ra possesses a leader sequence, 50% or more of this protein remains cell associated. Do these observations suggest that members of the IL-1 family possess important intracellular functions, as yet undetermined? IL-1 alpha may play an intracellular role in regulating senescence; an IL-1 alpha antisense oligodeoxynucleotide was shown to prolong the life span of cultured human endothelial cells. Whether intracellular IL-1ra plays a role in influencing life span has not been determined. The discovery of IL-1ra has led to a first level of assumptions that this molecule may be functioning in vivo to regulate the pleiotropic extracellular effects of IL-1 in physiological or pathophysiological processes. Although enticing, these assumptions have not yet been proven to be true. Perhaps we need to look beyond, or within, and consider that IL-1ra and other members of the IL-1 family may have additional roles in normal or abnormal cell growth and development.

Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon.

Abstract: Normal structure and function of the lung parenchyma depend upon elastic fibers. Amorphous elastin is biochemically stable in vitro, and may provide a metabolically stable structural framework for the lung parenchyma. To test the metabolic stability of elastin in the normal human lung parenchyma, we have (a) estimated the time elapsed since the synthesis of the protein through measurement of aspartic acid racemization and (b) modeled the elastin turnover through measurement of the prevalence of nuclear weapons-related 14C. Elastin purified by a new technique from normal lung parenchyma was hydrolyzed; then the prevalences of D-aspartate and 14C were measured by gas chromatography and accelerator-mass spectrometry, respectively. D-aspartate increased linearly with age; Kasp (1.76 x 10(-3) yr(-1) was similar to that previously found for extraordinarily stable human tissues, indicating that the age of lung parenchymal elastin corresponded with the age of the subject. Radiocarbon prevalence data also were consistent with extraordinary metabolic stability of elastin; the calculated mean carbon residence time in elastin was 74 yr (95% confidence limits, 40-174 yr). These results indicate that airspace enlargement characteristic of "aging lung" is not associated with appreciable new synthesis of lung parenchymal elastin. The present study provides the first tissue-specific evaluation of turnover of an extracellular matrix component in humans and underscores the potential importance of elastin for maintenance of normal lung structure. Most importantly, the present work provides a foundation for strategies to directly evaluate extracellular matrix injury and repair in diseases of lung (especially pulmonary emphysema), vascular tissue, and skin.

Expression of AT2 receptors in the developing rat fetus.

Abstract: Angiotensin II is known primarily for its effects on blood pressure and electrolyte homeostasis, but recent studies suggest that angiotensin II may play a role in the regulation of cellular growth. This study was undertaken to identify the angiotensin II receptor subtypes expressed during fetal and neonatal development and to characterize their cellular localization. Using an in situ receptor binding assay on sagittal frozen sections of fetal and neonatal rats, bound 125I-[Sar1,Ile8]-angiotensin II was visualized by film and emulsion autoradiography. Bound radioligand was detected by E11 (embryonic day 11) and maximal binding occurred by E19-21. Radioligand binding remained unaltered 30 min after birth, whereas a noticeable and stable decrease was observed 12 h postparturition. The highly abundant angiotensin II receptors were shown to be AT2 by the marked reduction in radioligand binding achieved with PD123177 (10(-7)M), a specific AT2 receptor antagonist, whereas DuP 753 (10(-5)M), an AT1 receptor antagonist, had little effect. Emulsion autoradiography showed radioligand binding in the undifferentiated mesenchyme of the submucosal layers of the intestine and stomach, connective tissue and choroid surrounding the retina, subdermal mesenchyme adjacent to developing cartilage, diaphragm, and tongue. Residual AT2 receptors were found on the dorsal subdermal region of the tongue 72 h after birth. AT1 receptors were detected in the placenta at E13 and in the aorta, kidney, lung, liver, and adrenal gland at E19-21, consistent with an adult distribution. The transient expression of AT2 receptors in the mesenchyme of the fetus suggests a role of angiotensin II in fetal development.

Production of transforming growth factor beta 1 during repair of arterial injury.

Abstract: Repair of arterial injury produced by balloon angioplasty leads to the formation of a neointima and a narrowing of the vascular lumen. In this study, we examined the possibility that smooth muscle cells (SMC) in injured rat carotid arteries are stimulated to produce type-1 transforming growth factor-beta (TGF-beta 1) during neointima formation in vivo. Levels of TGF-beta 1 transcripts (2.4 kb) were significantly increased within 6 h after carotid injury and reached a maximum (five to sevenfold) by 24 h. Regenerating left carotids had sustained increases in TGF-beta 1 mRNA levels (about fivefold) over the next 2 wk, during which time a substantial neointimal thickening was formed. No changes in basal TGF-beta 1 mRNA levels were found in contralateral uninjured carotids at any of the times examined. Immunohistochemical studies showed that a large majority of neointimal SMC were stained for TGF-beta 1 protein in an intracellular pattern, consistent with active TGF-beta 1 synthesis in this tissue. Neointima formation and TGF-beta 1 immunoreactivity were correlated with increases in fibronectin, collagen alpha 2(I), and collagen alpha 1(III) gene expression. Infusion of purified, recombinant TGF-beta 1 into rats with a preexisting neointima produced a significant stimulation of carotid neointimal SMC DNA synthesis. These results suggest that TGF-beta 1 plays an important role as an endogenous growth regulatory factor produced by neointimal SMC themselves during progressive neointimal thickening after balloon angioplasty.

Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.

Abstract: Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by LN-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions ofpotassium, verapamil, calcium-free medium, and antagonists of the Kc. channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses toother agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on microcarrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator, the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the Kc. channel) on the endothelial cell membrane that leads to the release ofnitric oxide. (J. Clin.

Effects of fat on insulin-stimulated carbohydrate metabolism in normal men.

Abstract: We have examined the onset and duration of the inhibitory effect of an intravenous infusion of lipid/heparin on total body carbohydrate and fat oxidation (by indirect calorimetry) and on glucose disappearance (with 6,6 D2-glucose and gas chromatography-mass spectrometry) in healthy men during euglycemic hyperinsulinemia. Glycogen synthase activity and concentrations of acetyl-CoA, free CoA-SH, citrate, and glucose-6-phosphate were measured in muscle biopsies obtained before and after insulin/lipid and insulin/saline infusions. Lipid increased insulin-inhibited fat oxidation (+40%) and decreased insulin-stimulated carbohydrate oxidation (-63%) within 1 h. These changes were associated with an increase (+489%) in the muscle acetyl-CoA/free CoA-SH ratio. Glucose disappearance did not decrease until 2-4 h later (-55%). This decrease was associated with a decrease in muscle glycogen synthase fractional velocity (-82%). The muscle content of citrate and glucose-6-phosphate did not change. We concluded that, during hyperinsulinemia, lipid promptly replaced carbohydrate as fuel for oxidation in muscle and hours later inhibited glucose uptake, presumably by interfering with muscle glycogen formation.

Role of T lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice.

Abstract: The role of CD4+ and CD8+ T lymphocytes in terminating respiratory syncytial virus (RSV) replication, causing disease, and protecting from reinfection was investigated using a BALB/c mouse model in which CD4+ or CD8+ lymphocytes or both were depleted by injections of Mab directed against the respective mouse lymphocyte determinants. Kinetics of RSV replication, illness, and pathology were assessed after primary infection and rechallenge. Both CD4+ and CD8+ lymphocyte subsets were involved in terminating RSV replication after primary infection. When both T lymphocyte subsets were depleted RSV replication was markedly prolonged, yet no illness was evident, suggesting that host immune response rather than viral cytocidal effect was the primary determinant of disease in mice. Both CD4+ and CD8+ lymphocytes contributed to illness, although CD8+ lymphocytes appeared to play the dominant role in this particular system. Analysis of histological responses suggested that CD4+ lymphocytes were required for the appearance of peribronchovascular lymphocytic aggregates seen in normal mice after rechallenge, and that the presence of alveolar lymphocytes was correlated with illness. It is postulated that antibody is an illness-sparing mechanism for protecting mice from RSV infection, and that T lymphocytes are an important determinant of illness. Further delineation of RSV-induced immunopathogenesis in primary infection and reinfection will provide important information for the development of vaccine strategies.

1,25-dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis.

Abstract: The hormone, 1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3), inhibits lymphocyte activation in vitro. We studied the ability of the vitamin D metabolite to interfere in vivo with a primary T cell-mediated model of autoimmunity, murine experimental autoimmune encephalomyelitis (EAE). Within 2 wk of antigenic challenge, immunized animals will develop acute paralysis with central nervous tissue inflammation. If mice survive, a rise in antibody titer develops within a month. The administration of 0.1 microgram 1,25-(OH)2-D3 i.p. given every other day for 15 d, starting 3 d before immunization, significantly prevented the development of EAE. The rise in antibody titer to myelin basic protein was also abrogated. Histopathologic lesions of EAE were inhibited by treatment with the sterol. These results suggest a potent immunosuppressive role for 1,25-(OH)2-D3 in vivo in the modulation of a cell-mediated model of autoimmunity.

L-arginine abrogates salt-sensitive hypertension in Dahl/Rapp rats.

Abstract: This study examined the contribution of nitric oxide (NO) to the susceptibility or resistance to the hypertensive effects of high sodium chloride (8.0% NaCl) intake in young Dahl/Rapp salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats. Using NG-monomethyl-L-arginine (L-NMMA) as a probe for NO production in vivo, we found that increasing dietary sodium chloride increased NO activity in salt-resistant rats, but not in salt-sensitive rats. Exogenous L-arginine, the substrate for NO synthesis, decreased blood pressure to normotensive levels in salt-sensitive rats made hypertensive for 2 wk from 8.0% NaCl chow. D-arginine had no effect on blood pressure of these rats and L-arginine did not change blood pressure of salt-resistant rats. Intraperitoneal injections of L-arginine and its precursor, L-citrulline, and oral L-arginine, but not D-arginine, prevented the increase in blood pressure in salt-sensitive rats on the high salt chow over 2 wk of observation. In contrast, L-arginine did not alter the development of hypertension in spontaneously hypertensive rats. Mean urinary cGMP levels were higher in salt-sensitive rats on oral L-arginine than salt-sensitive rats on D-arginine. Infusion of L-NMMA acutely decreased, whereas intravenous L-arginine rapidly increased, urinary cGMP in both groups. L-arginine and L-citrulline increased production of NO and prevented salt-sensitive hypertension in Dahl/Rapp rats.

Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation.

Abstract: Mesangial cell proliferation is common in glomerulonephritis but it is unclear if proliferation is associated with any in vivo alteration in phenotype. We investigated whether mesangial of mesangial proliferative nephritis induced with antibody to the Thy-1 antigen present on mesangial cells. At day 3 glomeruli displayed de novo immunostaining for alpha-smooth muscle actin in a mesangial pattern, correlating with the onset of proliferation, and persisting until day 14. An increase in desmin and vimentin in mesangial regions was also noted. Immunoelectron microscopy confirmed that the actin-positive cells were mesangial cells, and double immunolabeling demonstrated that the smooth muscle actin-positive cells were actively proliferating. Northern analysis of isolated glomerular RNA confirmed an increase in alpha and beta/gamma actin mRNA at days 3 and 5. Complement depletion or platelet depletion prevented or reduced proliferation, respectively; these maneuvers also prevented smooth muscle actin and actin gene expression. Studies of five other experimental models of nephritis confirmed that smooth muscle actin expression is a marker for mesangial cell injury. Thus, mesangial cell proliferation in glomerulonephritis in the rat is associated with a distinct phenotypic change in which mesangial cell assume smooth muscle cell characteristics.

Apoptosis as a mechanism of cell death in cultured T lymphoblasts acutely infected with HIV-1.

Abstract: The mechanisms by which HIV-1 infection kills T lymphocytes are not clearly established. Apoptosis is an internally programmed cell death pathway that may regulate both T cell development and senescence, and that is characterized by cleavage of DNA at internucleosomal regions. The present experiments show that acute HIV-1 infection of MT2 lymphoblasts and activated normal peripheral blood mononuclear cells induces apoptosis. The addition of anti-gp120 neutralizing antibody, after HIV-1 infection of MT2 cells, permitted sustained high levels of viral replication, but blocked apoptosis and cell death. Apoptosis may account for the direct cytopathologic effects of HIV-1 in T cells.

Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C.

Abstract: A possible relationship between protein kinase C activation and impaired receptor-mediated endothelium-dependent relaxation in diabetes mellitus was examined in isolated aorta from normal rabbit exposed to elevated glucose. Aorta treated for 10 min with 4-phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, showed decreased relaxations to the endothelium-dependent vasodilator, acetylcholine, similar to normal aorta exposed to elevated glucose (22 and 44 mM) for 6 h. Relaxations to the receptor-independent endothelium-dependent vasodilator, A23187, and those caused by the direct smooth muscle vasodilator, sodium nitroprusside, were unaffected by treatment with PMA or exposure to elevated glucose. Indomethacin increased relaxations to acetylcholine of aorta treated with PMA indicating a role for vasoconstrictor prostanoids. PMA caused a significant increase in basal and acetylcholine-stimulated release of vasoconstrictor prostanoids including thromboxane A2 from aortic segments with, but not without endothelium. Protein kinase C inhibitors, H-7 or sphingosine, restored the abnormal acetylcholine-induced relaxations as well as suppressed the abnormal release of prostanoids in aorta exposed to elevated glucose. These findings suggest that the dysfunction of receptor-mediated endothelium-dependent relaxation associated with exposure to elevated glucose is due to increased production of vasoconstrictor prostanoids by the endothelium as a consequence of protein kinase C activation.

Chemotactic factors regulate lectin adhesion molecule 1 (LECAM-1)-dependent neutrophil adhesion to cytokine-stimulated endothelial cells in vitro.

Abstract: Monoclonal antibodies recognizing CD18, CD11a, CD11b, and neutrophil lectin adhesion molecule 1 (LECAM-1), i.e., the human homologue of the murine MEL-14 antigen, were used to assess the relative contribution of these glycoproteins to neutrophil-endothelial adhesion. Under static conditions, the adhesion of neutrophils to IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers was inhibited by antibodies to CD18, CD11a, and the neutrophil LECAM-1, and the effect of combining anti-LECAM-1 and anti-CD11a was almost additive. Under flow at a wall shear stress 1.85 dyn/cm2, a condition where CD18-dependent adhesion is minimal, anti-LECAM-1 inhibited adhesion by greater than 50%. Chemotactic stimulation of neutrophils induced a rapid loss of LECAM-1 from the neutrophil surface, and the level of neutrophil surface LECAM-1 was closely correlated with adhesion under flow. Neutrophils contacting the activated endothelial cells for 30 min lost much of their surface LECAM-1, a phenomenon induced by a soluble factor or factors released into the medium by the stimulated monolayers, and a high percentage migrated through the HUVEC monolayer. This migration was almost completely inhibited by anti-CD18, but was unaffected by antibodies to neutrophil LECAM-1. These results support the concept that LECAM-1 is a neutrophil adhesion molecule that participates in the adherence of unstimulated neutrophils to cytokine-stimulated endothelial cells under conditions of flow, and is then lost from the neutrophil surface coincident with the engagement of CD18-dependent mechanisms leading to transendothelial migration.

Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine.

Abstract: Elevated levels of plasma homocysteine are associated with both venous and arterial thrombosis. Homocysteine inhibits the function of thrombomodulin, an anticoagulant glycoprotein on the endothelial surface that serves as a cofactor for the activation of protein C by thrombin. The effects of homocysteine on thrombomodulin expression and protein C activation were investigated in cultured human umbilical vein endothelial cells and CV-1(18A) cells that express recombinant human thrombomodulin. Addition of 5 mM homocysteine to endothelial cells produced slight increases in thrombomodulin mRNA and thrombomodulin synthesis without affecting cell viability. In both cell types, thrombomodulin synthesized in the presence of homocysteine remained sensitive to digestion with endoglycosidase H and failed to appear on the cell surface, suggesting impaired transit along the secretory pathway. In a cell-free protein C activation assay, homocysteine irreversibly inactivated both thrombomodulin and protein C in a process that required free thiol groups and was inhibited by the oxidizing agents diamide or N-ethylmaleimide. By inhibiting both thrombomodulin surface expression and protein C activation, homocysteine may contribute to the development of thrombosis in patients with cystathionine beta-synthase deficiency.