The effect of phosphodiesterase III inhibitors on human neutrophil function

OBJECTIVES: Neutrophils play an important role in ridding the body of bacteria and cellular debris. Several neutrophil functions are thought to be regulated by inotropes that increase cellular levels of cyclic adenosine monophosphate, including phosphodiesterase (PDE) inhibitors. We have investigated the effect of amrinone, milrinone, and olprinone, type III PDE (PDE-III) inhibitors, on several human neutrophil functions. DESIGN: Prospective in vitro study. SETTING: Academic research laboratory. SUBJECTS: Neutrophils isolated from 12 healthy adult volunteers. INTERVENTIONS: We measured chemotaxis, phagocytosis, reactive oxygen species production, intracellular calcium ion concentration, and cyclic adenosine monophosphate levels in neutrophils in the absence and the presence (at clinically relevant concentrations, 10 times, and 100 times those concentrations) of amrinone, milrinone, or olprinone. We also measured reactive oxygen species production under the same condition in a xanthine-xanthine oxidase system MEASUREMENTS AND MAIN RESULTS: None of the PDE-III inhibitors impaired neutrophil chemotaxis or phagocytosis. Amrinone at clinically relevant or higher concentrations and milrinone at high concentrations reduced superoxide, hydrogen peroxide, and hydroxyl radical levels in neutrophils and in the xanthine-xanthine oxidase system. Olprinone did not have those effects, and none of the PDE-III inhibitors had an effect on intracellular calcium ion concentration or cyclic adenosine monophosphate production in neutrophils stimulated by a chemotactic factor. CONCLUSIONS: The ability of amrinone to scavenge reactive oxygen species at clinically relevant concentrations while not affecting neutrophil function suggests that the PDE inhibitor can be used without detriment in severely ill patients.     

Endothelial and vascular smooth muscle function in sepsis

The vascular abnormalities that arise during sepsis imply disturbances in the delicately tuned homeostatic mechanisms of vascular endothelium and smooth muscle. In the microvasculature, smooth muscle tone represents a complex equilibrium among metabolic stimuli, hemodynamic forces, and neurohumoral influences. Local tissue perfusion also is modulated by vasoactive mediators that can be produced locally, through endothelium-dependent adhesion and activetion of inflammatory cells, or at a distance. In sepsis, derangement of normal autoregulation of perfusion, together with toxic effects of mediators, may be severe enough to result in organ dysfunction. Recent advances in vascular biology have illuminated a variety of targets, such as adhesion molecules, platelet activating factor, and inducible nitric oxide synthase for potential therapeutic intervention in sepsis.     

Decay-accelerating factor is expressed on vascular smooth muscle cells in human atherosclerotic lesions.

Decay-accelerating factor (DAF) is a constitutively expressed plasma membrane glycoprotein on blood cells and endothelium that inhibits cell surface C3/C5 convertase formation, thus inhibiting complement activation and protecting cells from lysis by the terminal complement components. Using monoclonal anti-DAF antibodies in conjunction with anti-smooth muscle cell (SMC)-specific myosin antibodies, it was found by immunohistochemistry that vascular SMC in advanced human carotid atherosclerotic lesions express DAF antigen. The percentage of DAF-positive SMC ranged from 20 to 60% between different patient samples and SMC DAF expression was limited to SMC in the lesion proper. Normal arterial wall SMC exhibited no DAF-specific immunostaining. Essentially 100% of passaged cultured vascular SMC derived from normal human uterine artery, or from umbilical vein, expressed DAF as assessed by immunocytochemistry. A 68-kD band was observed on SDS-PAGE autoradiograms of DAF-immunoprecipitated radiolabeled cultured SMC extracts. Sensitization of rabbit erythrocytes with DAF-containing SMC extracts conferred protection against complement-mediated hemolysis in normal human serum and the protective effect could be reversed by treatment with anti-DAF antibodies. We conclude that DAF is induced on vascular SMC during atherogenesis and in culture.     

Cirrhosis of the liver and receptor-mediated function in vascular smooth muscle

Altered regulation of receptors on the vascular smooth muscle has been proposed as one of the mechanisms that may account for the vascular abnormalities in patients with cirrhosis of the liver. Impaired contractility and down-regulation of contractile receptors have been demonstrated in cirrhotic patients and animal models, although interpretation of the literature is hampered by methodological variation and conflicting results. There is little evidence, however, that receptor down-regulation is the cause of contractile dysfunction in either patients or animal models. Receptor desensitisation may contribute to impaired contraction in human arteries, but further investigation is required to confirm this possibility.     

The alterations of vascular smooth muscle reactivity in vitro by human chorionic gonadotrophin

The study tests the hypothesis that human chorionic gonadotrophin (hCG) alters vascular smooth muscle reactivity by examining the effect of hCG administration on the contractility and relaxation of isolated vascular smooth muscle. Aortic rings from rats pre-treated with intraperitoneal administration of 5,000 I.U of hCG and control animals were contracted to phenylephrine, angiotensin II, CaCl₂ and KCl. The experiments with phenylephrine were repeated with rings that were either de-endothelialized, incubated with L-NMMA, or incubated with calcium ionophore A23187. Aortic rings precontracted with phenylephrine were relaxed to acetylcholine (endothelium-dependent), sodium nitroprusside, hydralazine (endothelium-independent) or in the presence of A23187. The contractile responses of aortic rings from hCG-treated animals to phenylephrine, angiotensin II, CaCl₂ and KCl were significantly attenuated. This effect was not reversed by pre-treatment with L-NMMA or by de-endothelialisation. In aortic rings from hCG-treated animals, there was almost total inhibition of acetylcholine-induced relaxation, but unaltered relaxation responses to sodium nitroprusside and hydralazine. The inhibitory effects of hCG-treatment on both the contraction and relaxation responses were either fully or partially reversed in the presence of calcium ionophore A23187. These observations suggest that hCG attenuates both contractile and endothelium-dependent relaxation responses by a mechanism which involves inhibition of extracellular calcium ion influx and may indicate a new role for the hormone in the altered vascular responses of both normal and abnormal pregnancies.     

Oxidative stress and vascular smooth muscle cell function in liver disease

Reactive oxygen species and reactive nitroxy species are now being recognized as regulatory molecules in signaling pathways influencing contractile and noncontractile functions of healthy vascular smooth muscle cells. In liver disease, oxidative stress is a systemic phenomenon, whose extent correlates with the severity of disease. A role for oxidative stress in the development of the hyperdynamic circulation in portal hypertension has been proposed. Evaluation of the limited available data indicates that it is premature to conclude that oxidative stress per se impacts on vascular smooth muscle cell function in liver disease.     

A novel vascular smooth muscle chymase is upregulated in hypertensive rats

While greater than 80% of angiotensin II (Ang II) formation in the human heart and greater than 60% in arteries appears to result from chymase activity, no cardiovascular cell-expressed chymase has been previously reported. We now describe the cloning of a full-length cDNA encoding a novel chymase from rat vascular smooth muscle cells. The cDNA encompasses 953 nucleotides, encodes 247 amino acids, and exhibits 74% and 80% homology in amino acid sequence to rat mast cell chymase I and II, respectively. Southern blot analysis indicates that the rat vascular chymase is encoded by a separate gene. This chymase was induced in hypertrophied rat pulmonary arteries, with 11-fold and 8-fold higher chymase mRNA levels in aortic and pulmonary artery smooth muscle cells from spontaneously hypertensive than in corresponding tissues from normotensive rats. We assayed the activity of the endogenous enzyme and of a recombinant, epitope-tagged chymase in transfected smooth muscle cells and showed that Ang II production from Ang I can be inhibited with chymostatin, but not EDTA or captopril. Spontaneously hypertensive rats show elevated chymase expression and increased chymostatin-inhibitable angiotensin-converting activity, suggesting a possible role for this novel enzyme in the pathophysiology of hypertension.     

Postjunctional vascular smooth muscle alpha-2 adrenoreceptors in human autonomic failure

Animal studies have suggested that vasoconstrictor alpha-2 adrenoreceptors exist on vascular smooth muscle cells. We tested this hypothesis in a patient with severe autonomic failure who demonstrated a pressor response to oral clonidine (a selective alpha-2 adrenoreceptor partial agonist). After clonidine 0.8 mg orally, mean arterial pressure rose by 54 mm Hg. After pretreatment with prazosin (a selective alpha-1 adrenoreceptor antagonist) and confirmation of alpha-1 blockade, clonidine 0.8 mg still raised mean arterial pressure by 43 mm Hg. After pretreatment with yohimbine (a selective alpha-2 adrenoreceptor antagonist), clonidine 0.8 mg elevated mean arterial pressure by 13 mm Hg. Since alpha-1 antagonism does not block, and alpha-2 antagonism does not block the pressor effect of clonidine, we conclude that clonidine raised blood pressure in this severely affected autonomic failure patient by vascular alpha-2 adrenoreceptor stimulation. Thus, this provides pharmacological evidence that postjunctional vascular smooth muscle alpha-2 adrenoreceptors exist in man and can modulate blood pressure.     

Endothelium-derived relaxing factor is a selective relaxant of vascular smooth muscle

The present study examines the relaxant selectivity of endothelium-derived relaxing factor (EDRF) released from cultured endothelial cells. Endothelial cells from bovine pulmonary artery (CCL-209) in culture were grown on Cytodex-3 microcarrier beads, packed into a column and superfused to release EDRF. EDRF response was estimated by its ability to relax phenylephrine-contracted rings of rabbit aorta. Bradykinin and A23187 (10(-10) to 10(-6) M) caused dose-dependent release of EDRF from cultured bovine pulmonary artery endothelial cells. The release was dependent on endothelial cell number. A23187 caused a larger and longer-lasting release of EDRF than bradykinin. EDRF relaxation was selective for blood vessels. EDRF relaxed rabbit aortic rings, but it did not relax histamine-contracted guinea pig tracheal, rabbit taenia coli strips or oxytocin-contracted guinea pig uterine rings. These nonvascular smooth muscles were, however, relaxed by isoproterenol (10(-4) M) and sodium nitroprusside (SNP, 10(-5) M). The sensitivity of guinea pig aortic rings and tracheal strips to SNP were compared. The IC50 values for SNP (10(-9) to 10(-5) M) were 0.07 and 0.3 microM for aortic rings and tracheal strips, respectively. Although the tracheal strips were about 4-fold less sensitive than the aorta toward SNP, a complete relaxation was achieved. These results suggest that EDRF relaxes vascular smooth muscles but not respiratory, Gl or reproductive smooth muscles. Thus, EDRF may be a selective relaxant of vascular smooth muscle.     

Inducible interleukin-1 gene expression in human vascular smooth muscle cells.

Interleukin-1 (IL-1) mediates many components of generalized host response to injury and may also contribute to local vascular pathology during immune or inflammatory responses. Because altered function of smooth muscle cells (SMC) accompanies certain vascular diseases, we tested whether SMC themselves might produce this hormone. Unstimulated SMC contain little or no IL-1 mRNA. However, exposure to bacterial endotoxin caused accumulation of IL-1 mRNA in SMC cultured from human vessels. Endotoxin maximally increased IL-1 beta mRNA in SMC after 4-6 h. The lowest effective concentration of endotoxin was 10 pg/ml. 10 ng/ml produced maximal increases in IL-1 beta mRNA. Interleukin-1 alpha mRNA was detected when SMC were incubated with endotoxin under "superinduction" conditions with cycloheximide. Endotoxin-stimulated SMC also released biologically functional IL-1, measured as thymocyte costimulation activity inhibitable by anti-IL-1 antibody. Thus, human SMC can express IL-1 beta and IL-1 alpha genes, or very similar ones, and secrete biologically active product in response to a pathological stimulus. Endogenous local production of this inflammatory mediator by the blood vessel wall's major cell type could play an important early role in the pathogenesis of vasculitis and arteriosclerosis.     

Specific binding of endothelin on human vascular smooth muscle cells in culture.

Endothelin is a newly discovered, potent vasoconstrictor peptide secreted by endothelial cells. The binding of endothelin was studied on cultured human vascular smooth muscle cells obtained from umbilical veins. A single specific binding site for 125I-endothelin was identified, with an apparent Kd of 126 pM and a maximal binding capacity of approximately 10,000 sites per smooth muscle cell. At room temperature the binding was saturable, reached equilibrium at 2 h (using 20 pM endothelin), and was slowly and only partially reversed by unlabeled endothelin. The calcium antagonists nifedipine, nicardipine, and diltiazem did not compete for the same binding site. Conditioned medium from cultured human umbilical vein endothelial cells inhibited the binding of 125I-endothelin dose dependently. This effect was antagonized by anti-endothelin antiserum. We conclude that human umbilical vein smooth muscle cells possess specific binding sites for endothelin, and that human endothelial cells secrete an endothelinlike material.     

Mechanical properties of vascular smooth muscle in hypertension

Reports from several laboratories indicate that the early part of smooth muscle contraction is achieved by normally cycling crossbridges, while the later part is subserved by slowly cycling or latch-bridges. We have recently found that the early bridges are responsible for almost 75% of the maximum shortening of the muscle. The latch bridges appear to be responsible for force production. The earliest change in the mechanical properties of caudal arteries from spontaneously hypertensive rats (SHR) is an increase in maximum shortening ability (delta Lmax), at a time when there is no detectable change in maximal isometric tetanic tension (Po). This substantiates the hypothesis that changes in Po are late indices of disease. The increased delta Lmax is associated with increased maximal velocity of shortening (Vo) of early cross-bridges, whereas latch bridge activity is normal. This provides the first subcellular explanation for the increase in delta Lmax. Since hypertension must result from narrowing of blood vessels, delta Lmax is, parenthetically, the important variable to study. Changes in Po, while contributing to increased vascular wall stiffness, do not directly account for the increased resistance. The cause of the increased cycling rate of crossbridges is probably increased myosin ATPase activity, or myosin light-chain phosphorylation by the specific kinase. Studies in helical sections of caudal, and segments of mesenteric resistance arteries provided similar results, confirming the suitability of caudal arteries as a model of resistance vascular units. The larger vessel is of course much easier to work with.     

Expression of class II transplantation antigen on vascular smooth muscle cells in human atherosclerosis

A large proportion of the cells of the human atherosclerotic plaque is assumed to be derived from medial smooth muscle cells. In contrast to these, the cells of the plaque have the capacity to accumulate lipid, and they also proliferate at a higher rate than medial cells. It has therefore been suggested that smooth muscle cells undergo a change of phenotype during atherogenesis, but there has been no evidence for such a change on the molecular level. We have now analyzed carotid artery plaques using a battery of antibodies against cell surface and cytoskeletal antigens, and found that most of the cells express the class II transplantation antigen (Ia antigen) HLA-DR. Also, the beta chain of HLA-DR was detected by immunoblotting of plaque extracts with the OKIa1 monoclonal antibody. HLA-DR is normally present on cells of the immune system, but only 60% of the DR-positive cells of the plaque reacted with monoclonal antibodies specific for macrophages and lymphocytes. Many of the remaining DR-positive cells contained the muscle-specific intermediate filament protein, desmin. This indicates that smooth muscle cells of atherosclerotic plaques express DR antigen. In contrast, very few DR-positive cells were found in normal human arteries. This suggests that expression of class II antigen is part of a phenotypic change in smooth muscle cells in atherosclerosis.     

Calcium antagonism in heart and vascular smooth muscle

Calcium antagonism is an unifying concept that offers a common denominator for a multitude of beneficial effects in cardiovascular therapy such as: direct reduction of myocardial energy expenditure and oxygen demand, improvement of myocardial oxygen supply resulting from spasmolytic and vasodilator effects on coronary trunk arteries (including collaterals and anastomoses), lowering of systemic arteriolar resistance (antihypertensive action), indirect diminition of cardiac oxygen requirement due to a fall in afterload, cardioprotection by prevention of excessive myocardial Ca uptake, damping effects on ectopic cardiac automaticity and on reentry pathways (not discussed in the present paper), and possibly anticalcinotic protection of the arterial walls at an advanced age or in cases of premature arterial senescence due to diabetes, hypertension, or nicotine intoxication.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype.

The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides.     

Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development

Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8-24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope-tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury.