Regulation of 12-lipoxygenase by cytokines in vascular smooth muscle cells

Increasing evidence suggests that cytokines such as interleukin-1beta (IL-1), IL-4, and IL-8 may play an important role in the chronic inflammation and cellular growth observed in cardiovascular diseases. The lipoxygenase (LO) pathway of arachidonate metabolism has also been related to the pathology of hypertension and atherosclerosis. LO products have chemotactic, hypertrophic, and mitogenic effects in vascular cells, and the LO enzyme has been implicated in the oxidation of LDL. Furthermore, earlier studies have shown that vascular smooth muscle cell (VSMC) growth factors such as angiotensin II and platelet-derived growth factor can increase LO activity and expression in VSMCs. In the present study, we have examined whether vasoactive and inflammatory cytokines such as IL-1, IL-4, and IL-8 can modulate 12-LO activity and expression in porcine VSMCs and also whether they have growth-promoting effects in these cells. Treatment of porcine VSMCs with these cytokines led to significant increases in the levels of a cell-associated 12-LO product, 12-hydroxyeicosatetraenoic acid, as well as intracellular 12-LO enzyme activity. Furthermore, each of these cytokines led to a dose-dependent increase in 12-LO mRNA expression (333-base pair PCR product) as well as 12-LO protein expression (72 kD). In addition, all three interleukins could induce significant increases in VSMC DNA synthesis as well as proliferation. These results suggest that these cytokines have mitogenic effects in VSMCs and are also potent positive regulators of the 12-LO pathway. Thus, enhanced 12-LO activity and expression may be a key mechanism for cytokine-induced VSMC migration and proliferation.     

Dipyridamole inhibits PDGF- and bFGF-induced vascular smooth muscle cell proliferation

Dipyridamole is the only pharmacologic agent demonstrated to reduce polytetrafluoroethylene (PTFE) graft occlusion in hemodialysis patients. However, the mechanism of action of dipyridamole in preventing graft occlusion is unknown. The purpose of this study was to examine the direct effects of dipyridamole on both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF)-induced vascular smooth muscle cell (VSMC) proliferation. Human aortic smooth muscle cells were grown to confluence in 96 well plates. A total of 5 x 10(-6) molar dipyridamole, PDGF 10 ng/ml, or bFGF 10 ng/ml were added to appropriate wells at the start of each experiment. Cell proliferation at 48 hours was determined using tritiated thymidine uptake. Intracellular cyclic AMP (cAMP) was measured using a competitive enzyme immunoassay. Treatment of VSMC with 5 microM dipyridamole dramatically reduced basal proliferation rates compared to controls [5229 +/- 1131 counts per minute (CPM) versus 387 +/- 68 CPM, P < 0.001]. Treatment with dipyridamole also reduced PDGF-stimulated VSMC proliferation (7311 +/- 1655 CPM vs. 593 +/- 110 CPM, P < 0.001) as well as the response to bFGF (5632 +/- 1270 CPM vs. 310 +/- 31 CPM, P < 0.001). Treatment of VSMC with either 5 or 20 microM dipyridamole did not change intracellular cAMP levels. Furthermore, the addition of dibutyryl cAMP to VSMC demonstrated only a modest inhibitory effect on proliferation. We conclude that dipyridamole inhibits both PDGF- and bFGF-stimulated VSMC proliferation. The effects of dipyridamole on VSMC proliferation do not appear to be entirely mediated by changes in intracellular cAMP concentrations. The direct effect of dipyridamole on VSMC proliferation may account for its efficacy in reducing PTFE graft thrombosis in hemodialysis patients.     

Hypercortisolemia increases plasma interleukin-10 concentrations during human endotoxemia--a clinical research center study

Hypercortisolemia directly before the administration of endotoxin (LPS) to normal humans completely prevents the release of the proinflammatory cytokine tumor necrosis factor, whereas hypercortisolemia 12 h to 7 days before the injection of LPS is associated with enhanced tumor necrosis factor release. To determine the effect of elevated cortisol levels on the secretion of the antiinflammatory cytokine interleukin-10 (IL-10), 23 healthy men were given iv LPS (lot EC-5; 2 ng/kg) alone or in combination with a continuous iv infusion of hydrocortisone (3 micrograms/kg.min) for 6 h immediately before or 6, 12, or 144 h before LPS injection. LPS induced a monophasic increase in plasma IL-10 concentrations that peaked after 2 h (162 +/- 27 pg/mL; P < 0.0001). In subjects who were infused with hydrocortisone directly before LPS administration, IL-10 concentrations were much higher (1784 +/- 331 pg/mL; P < 0.0001 vs. LPS only), whereas hypercortisolemia 6, 12, or 144 h before LPS injection did not influence LPS-induced IL-10 levels. In human whole blood in vitro, hydrocortisone caused a dose-dependent reduction of LPS-induced IL-10 levels. Further, hydrocortisone reversed the increase in IL-10 concentrations by epinephrine in LPS-stimulated whole blood. Stimulation of IL-10 release may contribute to the antiinflammatory properties of glucocorticoids.     

cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway

BACKGROUND: Abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the pathogenesis of atherosclerosis and many vascular diseases. It is known that nitric oxide released from the endothelium participates in the regulation of VSMC proliferation via a cyclic 3',5'-guanosine monophosphate (cGMP)-mediated mechanism. In a series of experiments, sodium nitroprusside (SNP) and A02131-1 were evaluated for their antiproliferative effect and the mechanism of their cGMP-elevating action. METHODS AND RESULTS: The effect of SNP and A02131-1 on epidermal growth factor (EGF)-stimulated proliferation of rat aortic smooth muscle cells (VSMC) was examined. Cell proliferation was measured in terms of [3H]thymidine incorporation, flow cytometry, and the cell number. Further, their effect on the EGF-activated signal transduction pathway was assessed by measuring mitogen-activated protein kinases (MAPK), MAPK kinase (MEK). Raf-1 activity, and the formation of active form of Ras. SNP and A02131-1 inhibited EGF-induced DNA synthesis and subsequent proliferation of VSMC. These two increased cGMP but only a little cAMP in VSMC. A similar antiproliferative effect was observed with 8-bromo-cGMP. The antiproliferative effect of the two was reversed by KT5823 but not by dideoxyadenosine nor Rp-cAMPS. SNP and A02131-1 blocked the EGF-inducible cell cycle progression at the G1/S phase. Further experiments indicated that the two cGMP-elevating agents primarily blocked the activation of Raf-1 by EGF-activated Ras. CONCLUSIONS: These results demonstrate that cGMP-elevating agents inhibit [3H]thymidine incorporation and thus the growth of VSMC, and this inhibition appears to attenuate EGF-activated signal transduction pathway by preventing Ras-dependent activation of Raf-1.     

Brown tumor of the mandible

OBJECTIVE: To investigate the inhibitory effect of IL-10 on inflammatory reaction in rat mesangial cells (rMC). METHODS: Cell proliferation was tested by 3H-thymidine uptake and absolute cell counts. The production of IL-1 and TNF alpha by rMC was assessed by bioactivity assay and their gene expression by Northern blot hybridization. The expression of intercellular adhesion molecule-1 (ICAM-1) on rMC was determined by ELISA. RESULTS: IL-10 (25 ng/ml) inhibited 5% FCS and IL-1 induced cell proliferation by 39% and 52% respectively. It also suppressed the production of IL-1 and TNF alpha bioactivity by rMC by 48% and 68%, which was consistent with the decline of IL-1 and TNF alpha gene expression. The IL-1-induced expression of ICAM-1 on surface of rMC was attenuated by IL-10 treatment. CONCLUSION: Our data suggest that IL-10 may be an inhibitory cytokine in regulation of inflammatory reaction in glomerular mesangial cells.     

Some experiences with hospital-based psychiatric aftercare over ten years

Smooth muscle cells represent a significant percentage of the total cells in the airway but their contribution to the inflammatory response seen in airway disease has not been studied. Hence, we have looked at the release of the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in response to bacterial lipopolysaccharide (LPS) and the pro-inflammatory cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Human airway smooth muscle (HASM) cells released GM-CSF under basal conditions (45.4 +/- 13.1 pg ml-1) that was significantly enhanced by IL-1 beta and TNF alpha with a maximal effect seen at 10 ng ml-1 (1.31 +/- 0.07 ng ml-1 and 0.72 +/- 0.16 ng ml-1, respectively). In contrast, neither LPS nor IFN gamma produced a significant increase in GM-CSF release. However, HASM cells exposed to IL-1 beta, TNF alpha and IFN gamma generated more GM-CSF than that evoked by any cytokine alone (2.2 +/- 0.15 ng ml-1). The release of GM-CSF elicited by the cytokine mixture was inhibited by cycloheximide and dexamethasone. These data suggest that HASM cells might play an active part in initiating and/or perpetuating airway inflammation in addition to controlling airway calibre.     

Diethyldithiocarbamate ameliorates the effect of lipopolysaccharide on both increased nitrite production by vascular smooth muscle cells and decreased contractile response of aortic rings

The depression of vasoconstrictor responsiveness caused by bacterial lipopolysaccharide (LPS) is mediated, in part, by the induction of nitric oxide synthase (NOS) and the resultant increase in nitric oxide production by vascular smooth muscle. The present study evaluated the ability of the antioxidant, diethyldithiocarbamate (DDTC), to attenuate the LPS-stimulated induction of NOS in cultured vascular smooth muscle cells (VSMC) and the depression of in vitro vascular reactivity caused by LPS administration to rats. The LPS-stimulated increase in nitrite production by cultured VSMC was inhibited 85% by DDTC (100 microM). When VSMC were stimulated with a combination of LPS, interferon-gamma (INF) and tumor necrosis factor (TNF) nitrite production was 5-fold greater than with LPS alone. DDTC inhibited 49% of the increase caused by LPS plus INF and TNF. Aortic rings taken from animals injected with LPS showed a depression of maximum force in response to phenylephrine which was reversed by inhibition of NOS activity. Pretreatment of animals with DDTC attenuated this depression of vascular reactivity. The DDTC treatment did not reduce the increase in serum TNF levels caused by LPS. These results suggest that DDTC can attenuate the LPS-stimulated induction of NOS in vascular smooth muscle and may thereby ameliorate the impairment of vascular reactivity.     

Ascorbate affects proliferation of guinea-pig vascular smooth muscle cells by direct and extracellular matrix-mediated effects

Proliferation of smooth muscle cells and deposition of extracellular matrix proteins are important events in the formation of atherosclerotic plaques. We have investigated the direct and matrix-mediated effects of ascorbate on the proliferation rate of vascular smooth muscle cells (VSMC) isolated from the guinea-pig aorta. In the presence of ascorbate, cells showed a bi-phasic growth pattern. At 125 microM ascorbate, -3H--thymidine incorporation was stimulated 25%. However, higher concentrations of ascorbate gradually decreased cell-incorporated radioactivity up to 50% at 2 mM ascorbate. These effects of ascorbate on DNA synthesis in VSMC were paralleled by the changes in cell number and were not due to ascorbate cytotoxicity. Alpha-tocopherol (0.1 mM), individually and in combinations with 1 mm ascorbate, also inhibited DNA synthesis in VSMC. Ascorbate also influenced proliferation of smooth muscle cells through matrix-mediated effect. New VSMC culture plated on extracellular matrices deposited by smooth muscle cells in the presence of 0.1-1 mM ascorbate had up to 50% lower proliferation rate than on matrices from ascorbate-deficient cells, as assessed by [3H]-thymidine incorporation. This effect was independent from alpha-tocopherol and specific inhibitors of collagen synthesis: L-azetidine-2-carboxylic acid and pyridine-2,4-dicarboxylic acid. An ascorbate-dependent matrix effect was specific for smooth muscle cells grown on VSMC and human skin fibroblast-originated matrices, but not for human vascular endothelial cells. The possible involvement of ascorbate in the regulation of smooth muscle cells proliferation by its antioxidant/pro-oxidant effects and regulation of extracellular matrix composition are discussed.     

Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells

OBJECTIVE: While natriuretic peptides can inhibit growth of vascular muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth. METHODS: Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein. RESULTS: In rat VSMC, passages 4-12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR. CONCLUSION: Our findings indicate that the guanyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanyl-cyclase-linked receptors.     

Cytokine-induced inhibition of bone matrix proteins is not mediated by prostaglandins

Interleukin-1 (IL-1) and tumor necrosis factor (TNF), two pleiotropic cytokines produced in inflammatory processes, inhibit bone matrix biosynthesis and stimulate prostanoid formation in osteoblasts. In the present study, the importance of prostaglandin formation in IL-1 and TNF-induced inhibition of osteocalcin and type I collagen formation has been examined. In the human osteoblastic cell line MG-63, IL-1 alpha (10-1000 pg/ml), IL-1 beta (3-300 pg/ml) and TNF-alpha (1-30 ng/ml) stimulated prostaglandin E2 (PGE2) formation and inhibited 1,25(OH)2-vitamin D3-induced osteocalcin biosynthesis as well as basal production of type I collagen. Addition of PGE2 or increasing the endogenous formation of PGE2 by treating the cells with arachidonic acid, bradykinin, Lys-bradykinin or des-Arg9-bradykinin, did not affect osteocalcin and type I collagen formation in unstimulated or 1,25(OH)2-vitamin D3-stimulated osteoblasts. Four non-steroidal antiinflammatory drugs, indomethacin, flurbiprofen, naproxen and meclofenamic acid, inhibited basal, IL-1 beta- and TNF-alpha-stimulated PGE2 formation in the MG-63 cells without affecting IL-1 beta- or TNF-alpha-induced inhibition of osteocalcin and type I collagen formation. In isolated, non-transformed, human osteoblast-like cells, IL-1 beta and TNF-alpha stimulated PGE2 formation and concomitantly inhibited 1,25(OH)2-vitamin D3-stimulated osteocalcin biosynthesis, without affecting type I collagen formation. In these cells, indomethacin and flurbiprofen abolished the effects of IL-1 beta and TNF-alpha on prostaglandin formation without affecting the inhibitory effects of the cytokines on osteocalcin biosynthesis. These data show that IL-1 and TNF inhibit osteocalcin and type I collagen formation in osteoblasts independently of prostaglandin biosynthesis and that non-steroidal antiinflammatory drugs do not affect the effects of IL-1 and TNF on bone matrix biosynthesis.     

Cytoprotective effects of adrenomedullin in glomerular cell injury: central role of cAMP signaling pathway

Activation of cAMP signaling pathway was shown to inhibit some pathobiologic processes in mesangial cells (MC). We investigated whether adrenomedullin (ADM), a potent agonist of adenylate cyclase, is synthesized in MC and whether it can, via cAMP, suppress the generation of reactive oxygen metabolites (ROM) and proliferation of cells in glomeruli. With the use of an immunohistologic technique ADM was detected in mesangial and microvascular areas of rat glomeruli. MC grown in primary culture synthesized ADM, and the synthesis was stimulated by TNF alpha and IL-1 beta but not by PDGF and EGF. ADM inhibited ROM generation in MC dose-dependently and caused in situ activation of protein kinase A (PKA). In macrophages (cell line J774) ROM generation was about four times higher than in MC and was inhibited by ADM in a similar way as in MC. The rate of MC proliferation, measured by [3H]-incorporation, and the activity of mitogen-activated protein kinase (MAPK) stimulated by PDGF and EGF were dose-dependently inhibited by ADM; the maximum inhibition (at 10 nM ADM) was about -80%. Mitogenesis of MC and MAPK activity when stimulated to a similar extent by endothelin (ET-1) was inhibited by ADM to a significantly (P < 0.01) lesser degree (-30%). Further, ADM inhibited PDF-stimulated mitogenesis and activation of MAPK in cultured vascular smooth muscle cells (VSMC). The inhibition of PDGF-activated MAPK by ADM in VSMC was reversed by the protein kinase A (PKA) inhibitor, H89. Taken together, results indicate the adrenomedullin (ADM) generated in mesangial cells (MC) can suppress, via activation of the cAMP-protein kinase A (PKA) signaling pathway, reactive oxygen metabolites (ROM) generation in MC and infiltrating macrophages as well as mitogen-activated protein kinase (MAPK)-mediated mitogenesis in MC and vascular smooth muscle cells (VSMC). We suggest that introglomerular ADM may serve as a cytoprotective autoacoid that suppresses pathobiologic processes evoked by immuno-inflammatory injury of glomeruli.     

A calcium influx is neither strictly associated with nor necessary for exocytotic membrane fusion in Paramecium cells

Alveolitis of sarcoidosis is characterized by activated alveolar macrophages (AMs) and T cells. The mediators interleukin-1 (IL-1) and interleukin 6 (IL-6) released by AMs represent essential factors for the progression of the T cells in the cell cycle. The role of IL-1 in pulmonary sarcoidosis has previously been studied; however, the relevance of other mediators (i.e. IL-6) has not yet been evaluated. We measured the spontaneous and lipopolysaccharide (LPS)-induced release of IL-6 and tumor necrosis factor alpha (TNF alpha) by bronchoalveolar lavage cells (BAL) and peripheral blood mononuclear cells (PBMNC) in 6 control subjects (group A) and in 15 patients with sarcoidosis, 10 with active (group B), 5 with inactive disease (group C). IL-6 as well as TNF alpha were spontaneously released by BAL cells of the active group in significantly greater amounts compared to both other groups; IL-6: A, 165.5 pg/ml/24 hr/10(6) cells (range, 0-604), B, 946 (0-2467), C, 16.6 (0-83); TNF alpha: A, 162 pg/ml/24 hr/10(6) cells (0-523), B, 803 (100-17352), C, 100 (0-379). In all groups autologous PBMNC proved to be quiescent, releasing only baseline levels of the cytokines tested. After stimulation with LPS all these cells released great quantities of IL-6 and TNF alpha. In active disease a positive correlation between IL-6 and TNF alpha release was observed (r = 0.77, p < 0.02). The present study documents that in active sarcoidosis the spontaneous release of IL-6 by BAL cells parallels the spontaneous release of TNF alpha. IL-6 is capable of initiating the proliferation and activation of T cells in the lung.     

Measuring the quality of outpatient treatment for schizophrenia

OBJECTIVE: Usually it is not possible to study the initial systemic response in patients with acute pancreatitis in the first hours after onset of the disease. We used postendoscopic retrograde pancreatography (ERP) pancreatitis as a model to study cytokine and anticytokine release in the early phase of human acute pancreatitis. METHODS: Post-ERP pancreatitis was defined as a threefold increase in serum amylase and at least two of the following clinical symptoms: abdominal pain, nausea, vomiting or peritonism 24 h after ERP. Serum levels of pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor alpha (TNF), as well as endogenous antagonistic mediators of the systemic inflammatory response such as soluble tumour necrosis factor alpha receptors p55 (TNFR p55) and p75 (TNFR p75), and IL-1-receptor antagonist (IL-1-RA) and interleukin-2-receptor (IL-2R) as indicators of lymphocyte activation were measured before and 0, 1, 4, 12, 24 and 48 h after ERP. In nine patients with acute post-ERP pancreatitis, these parameters were monitored daily until C-reactive protein (CRP) was within normal ranges and were compared to patients without pancreatitis after ERP. RESULTS: IL-1beta was not detectable in five patients with and four patients without post-ERP pancreatitis. The values of the remaining patients in both groups were lower than 3.9 pg/ml. IL-8 and IL-1-RA serum concentrations peaked 12 h after ERP (132.9 and 3245.0 pg/ml respectively) compared to patients without post-ERP pancreatitis (25.8 and 389.9 pg/ml respectively). The IL-6 concentration increased to 81.6 pg/ml (8.0 pg/ml in control patients) 24 h after ERP, while the peak values for CRP were measured 72 h after ERP (164.0 versus 7.7 mg/l). IL-2R content was maximally elevated 144 h after ERP (688.8 versus 255.9 U/ml), while concentrations of TNF and its receptors showed no significant change over time. CONCLUSION: The initial response of the cytokine network to damage of the human pancreas leading to acute pancreatitis includes the release of IL-8 and the IL-1 antagonist IL-1-RA, while IL-1beta is not found in the systemic circulation. The TNF system does not seem to be involved as indicated by the lack of detectable changes in TNF and the soluble TNFR p55 and p75 serum concentrations. Lymphocyte activation as indicated by elevated IL-2R levels occurred days after the initial trauma. Even mild post-ERP pancreatitis leads to significant systemic release of cytokines and their biological counterparts.     

Cytokines in vitreous humor: interleukin-6 is elevated in proliferative vitreoretinopathy

PURPOSE: To measure the levels of interleukins (IL) 1 beta, 6, and 8, and tumor necrosis factor-alpha (TNF alpha) in the vitreous of patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), vitreous hemorrhage, and macular pucker. METHODS: Vitreous samples were collected, undiluted, from patients with PVR, PDR of varying severity, and miscellaneous lesions (vitreous hemorrhage from trauma, macular degeneration, vein occlusion, and non-PVR patients with giant tear, retinal detachment, and macular pucker). Immunoreactive levels of the cytokines, IL-1 beta, IL-6, IL-8, and TNF alpha were determined by enzyme-linked immunoadsorbent assays, and samples were analyzed for protein and hyaluronic acid content using standard assays. RESULTS: The levels of TNF alpha were below detection limits of the assay (< 3 pg/ml). In 45 of the 47 samples tested, IL-1 beta levels also were below detection limits of the assay (< 3 pg/ml). IL-6 levels ranged from < 30 to 5487 pg/ml, with the highest values observed in the PVR patients. IL-8 levels ranged from < 20 to 1900 pg/ml, and were consistently high in the miscellaneous group. Some of the PVR patients with C2 and C3 level severity also exhibited IL-8 levels exceeding 100 pg/ml. In a second study, IL-6 content of vitreous from miscellaneous and PVR patients was compared. In this study, significantly elevated levels of IL-6 were observed in the PVR patients (91.5 +/- 18 pg/ml) compared to the miscellaneous group (10.3 +/- 3.7 pg/ml) CONCLUSIONS: Elevated levels of IL-6 in the vitreous occur in PVR, implicating a role for this cytokine in the pathogenesis of this ocular disorder.     

Clinical pharmacokinetics of irinotecan

Nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) have been reported to prevent vascular smooth muscle cell (VSMC) proliferation and have beneficial effects to reduce intimal thickening in response to arterial injury. The purpose of this study was to determine whether the downstream effector molecule of NO-cGMP signaling, cyclic GMP-dependent protein kinase (PKG), regulates phenotypic modulation and proliferation in cultured rat aortic VSMC. PKG-expressing VSMC lines were created by transfection of PKG-deficient cell lines and characterized. All forms of PKG, i.e. PKG-I alpha and PKG-I beta, as well as the constitutively active catalytic domain of PKG-I, transformed dedifferentiated 'synthetic' VSMC to a more contractile-like morphology. PKG expression resulted in an increased production of the contractile phenotype marker proteins, smooth muscle myosin heavy chain-2, calponin and alpha-actin and restored the capacity of cAMP and cGMP analogues to inhibit platelet-derived growth factor (PDGF)-induced cell migration. On the other hand, PKG expression had no significant effects on PDGF-induced cell proliferation. These results suggest that PKG expression contributes to the regulation of a contractile-like phenotypic expression in cultured VSMC, and the suppression of PKG expression during cultured growth in vitro may permit the modulation of cells to a more synthetic, dedifferentiated phenotype.     

Soluble cytokine receptors and receptor antagonists are sequentially released after trauma

Cytokine receptors and receptor antagonists (RAs) have been identified in trauma patients. We hypothesized that after traumatic injury, a sequential release of soluble cytokine receptors and RAs may exist that mirrors the release of the primary cytokines themselves. Twenty-two patients were included in the study: 14 males and 8 females. The mean age was 30.1 +/- 12.5 (range, 19 to 71), and the mean Injury Severity Score was 28.7 +/- 12.6 (range, 4 to 57). There were 15 survivors and 7 nonsurvivors. Samples were collected on arrival to the emergency department and at serial intervals for up to 7 days. Monoclonal antibody enzyme-linked immunosorbent assay kits to tumor necrosis factor (TNF), soluble TNF-receptor (sTNF-R) 55 kd and 75 kd, interleukin (IL)-1 and IL-1 RA, and IL-2 and IL-2r were used. Sera from 22 healthy individuals were used as normal controls. No TNF, IL-1, or IL-2 could be detected in any patient sera after injury. Control levels for the soluble cytokine receptors and RAs were as follows: sTNF-R 55 kd, 607 +/- 89 pg/mL; sTNF-R 75 kd, 2,141 +/- 169 pg/mL; IL-1 RA, 291 +/- 35 pg/mL; and IL-2r, 426 +/- 53 U/mL. In trauma patients, both 55 kd and 75 kd sTNF-R were significantly elevated on arrival to the emergency department, with values of 2,441 +/- 506 pg/mL (p < 0.001) and 4,736 +/- 537 pg/mL (p < 0.001), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells

BACKGROUND: Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. METHODS AND RESULTS: We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. CONCLUSIONS: The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.