Nonmuscle myosin heavy chain-B expression in balloon-dilated and stented arteries: a study in the atherosclerotic Yucatan micropig.

BACKGROUND: Restenosis after balloon angioplasty is in part due to remodelling, whereas restenosis after stenting is entirely due to neointima formation. Nonmuscle myosin heavy chain-B (NMMHC-B) is expressed by vascular smooth muscle cells and because of its overexpression in restenotic lesions after balloon angioplasty, NMMHC-B is proposed as a potential therapeutic target. Because the mechanisms underlying restenosis after balloon angioplasty or after stenting are different we hypothesised that the expression of NMMHC-B would differ in balloon-dilated versus stented arteries. METHODS: To study the localisation and time course of expression of NMMHC-B, we performed stenting or balloon dilation in peripheral arteries of 16 atherosclerotic Yucatan micropigs and used serial intravascular ultrasound (IVUS) and angiography to measure geometric dimensions following balloon angioplasty or stenting. In situ hybridisation techniques were used to detect NMMHC-B mRNA. 5'-bromo-2'-deoxyuridine (BrdU) was administered to detect proliferating cells. By counting the number of silver grains in the different layers of the artery, we could compare the amount of expression at the different time points between the groups. RESULTS: In intima and media, NMMHC-B expression increased after balloon dilation and stenting and peaked at 7 days. In stented arteries, the expression of NMMHC-B remained high for up to 42 days after injury, whereas in balloon-dilated arteries it had normalised. In the adventitia of balloon-dilated arteries, but not of stented arteries, NMMHC-B expression peaked at 7 days. NMMHC-B expression was not limited to proliferating cells. CONCLUSION: NMMHC-B is expressed near sites of active repair after arterial injury, but not limited to proliferating cells. The different pattern of NMMHC-B expression after balloon dilation compared with stenting may be related to arterial remodelling, because stented arteries that do not remodel lack this conspicuous adventitial expression at 7 days.

Adventitial cells do not contribute to neointimal mass after balloon angioplasty of the rat common carotid artery.

BACKGROUND: Previous studies suggest that the migration of adventitial cells into the neointima after balloon angioplasty might have an important role in vascular lesion formation. The current experiments were designed to study the migration of adventitial cells in response to mechanical injury of the rat carotid artery. METHODS AND RESULTS: Adventitial cells were stained in situ with PKH26, a fluorescent dye, after balloon angioplasty of the rat common carotid artery. Animals were killed at different time points, and tissue sections were examined under light and fluorescence microscopy. PKH26-labeled cells were detected exclusively in the adventitia. No labeled cells were present in the media or the neointima at any time point examined. A highly cellular neoadventitial layer composed of myofibroblasts exhibited an extensive proliferative response 3 days after injury over the entire adventitial circumference. CONCLUSIONS: Despite the prominent role that adventitial myofibroblasts seem to have in the postangioplasty remodeling process, they do not migrate to the medial or intimal layers in the rat carotid artery angioplasty model.

Perivascular inflammation after balloon angioplasty of porcine coronary arteries.

BACKGROUND: Inflammation has been suggested to play a role in vascular lesion formation after angioplasty. Whereas previous studies have focused on inflammatory reactions in the intima and media, less attention has been paid to adventitial and perivascular responses and their potential role in vascular remodeling. METHODS AND RESULTS: Balloon overstretch injury of porcine coronary arteries was performed with standard clinical angioplasty catheters. Vessels were examined from 0.5 hour to 14 days after injury by immunohistochemistry and in situ hybridization (ISH) for neutrophil and macrophage markers, cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule-1), and neutrophil-specific CXC chemokines (alveolar macrophage-derived neutrophil chemotactic factor [AMCF]-I/interleukin-8 and AMCF-II). Neutrophils accumulated in the adventitia surrounding the injury site from 2 hours to 3 days, followed by macrophages from 1 to 7 days after angioplasty. Inflammation was associated temporally with the expression of mRNAs encoding cell adhesion molecules and chemokines. The main inflammatory and proliferative foci were not limited to the adventitia but rather extended many millimeters away from the injured vessel throughout the surrounding adipose and myocardial tissues. CONCLUSIONS: Inflammatory responses after angioplasty of porcine coronary arteries occurred throughout the entire perivascular tissue. We hypothesize that perivascular inflammatory cells play a role in the recruitment and/or proliferation of adventitial myofibroblasts, possibly through the release of reactive oxygen species and/or cytokines, and thus contribute to vascular remodeling associated with postangioplasty restenosis.

Inhibition of vascular cell growth by X-ray irradiation: comparison with gamma radiation and mechanism of action.

PURPOSE: Catheter-based delivery of gamma and beta radiation effectively inhibits restenosis. Major disadvantages of these radioisotopes include continuous emission; excessive depth of penetration, creating safety hazards (gamma); and inadequate penetration, limiting effectiveness (beta). Low-voltage X-rays have a distinct potential advantage, because the source is active only when current is applied, and depth of penetration is voltage dependent. This study was performed to determine if low-voltage X-rays inhibit smooth muscle and adventitial cell growth in vitro and to determine the molecular mechanisms involved in this cellular response. METHODS AND RESULTS: Vascular cells in culture were exposed to low-voltage X-ray radiation and analyzed for their subsequent ability to proliferate. X-ray irradiation caused a dose-dependent inhibition in proliferation, similar to the effect seen with equivalent doses of gamma radiation. The radiation-induced inhibition of proliferation did not appear to be related to apoptosis, but rather to delayed progression through the cell cycle, because a 65% increase in the proportion of cells in S phase was seen 24-96 h after X-ray exposure compared to control. Expression of p53, a cell cycle transcriptional activator, and p21, a cell cycle inhibitor, were significantly elevated after exposure to low-voltage X-rays, providing a potential mechanism for this delay. CONCLUSIONS: Low-voltage X-rays can effectively inhibit proliferation of vascular smooth muscle and adventitial cells. This inhibition is apparently due to a delay in progression through the cell cycle, which is mediated by increases in the levels of cell cycle inhibitors.

Localization of human ATP-binding cassette transporter 1 (ABC1) in normal and atherosclerotic tissues.

The present study examines the expression of ATP-binding cassette transporter 1 (ABC1) mRNA in normal and atherosclerotic tissues by using in situ hybridization in an effort to better understand the function of this cholesterol transport protein. Samples of normal baboon tissues as well as human normal and atherosclerotic aortas were hybridized with (35)S-labeled ABC1 sense and antisense riboprobes. Widespread expression of ABC1 was observed generally in tissues containing inflammatory cells and lymphocytes. Other noninflammatory cells that were also sites of ABC1 synthesis included the ductal cells of the kidney medulla, Leydig cells in the testis, and glial cells in the baboon cerebellum. Although normal veins and arteries did not express ABC1 mRNA, it was found to be upregulated in the setting of atherosclerosis, where widespread expression was found in macrophages within atherosclerotic lesions. These results are consistent with the proposed role of ABC1 in cholesterol transport in inflammatory cells. The specific upregulation of ABC1 mRNA in the setting of atherosclerosis probably reflects the response of leukocytes to cholesterol loading. However, the presence of ABC1 in ductal cells of the kidney medulla and in the small intestine suggest a more general role for this protein in cholesterol transport in other cell types.

Adventitial remodeling after angioplasty is associated with expression of tenascin mRNA by adventitial myofibroblasts.

OBJECTIVES: The purpose of this study was to determine the temporospatial expression of tenascin-C (TnC) in balloon-injured rat and porcine arteries. BACKGROUND: Recent studies suggest that cell migration, in addition to cell proliferation, is a critical component of neointima formation after vascular injury. We have previously shown that adventitial myofibroblasts synthesize growth factors that contribute to the formation of neointima after arterial injury. We have also shown that the extracellular matrix protein, TnC, regulates cell migration. Consequently, we investigated the temporospatial expression of TnC by myofibroblasts after vascular injury. METHODS: In situ hybridization and immunohistochemistry were used to investigate the temporospatial expression of TnC in injured arteries. Northern and Western blots were used to determine the in vitro expression of TnC. RESULTS: In situ hybridization revealed that the major site of TnC expression early after vascular injury was the adventitial myofibroblasts. Immunohistochemical staining demonstrated that TnC expression began in adventitial myofibroblasts three days after injury. Tenascin-C expression, however, did not persist in this region. Rather, it moved progressively across the vascular wall toward the luminal surface. By one week, TnC expression reached the developing neointima. In vitro, myofibroblasts did not express TnC mRNA under basal conditions. In contrast, angiotensin II and PDGF-BB, factors that have been implicated in remodeling of balloon-injured arteries, markedly upregulated TnC mRNA. CONCLUSIONS: Tenascin-C is expressed in response to balloon injury. Tenascin-C expression begins with adventitial myofibroblasts. Over a period of 7 to 14 days, expression moves progressively across the vessel wall to the neointima. We hypothesize that adventitial myofibroblasts are actively involved in the formation of neointima and that TnC facilitates migration of these cells during adventitial remodeling.

Characterization of epithelial chemoattractants for human intestinal intraepithelial lymphocytes.

BACKGROUND & AIMS: Although homing of intraepithelial lymphocytes (IEL) into intestinal epithelia seems to be guided by signals from epithelia, little is known concerning functional epithelial-derived chemoattractants for IEL. METHODS: Epithelial chemoattractants for IEL were analyzed using chemotaxis chamber system, enzyme-linked immunosorbent assay, and in situ hybridization using human epithelial lines and IEL lines. RESULTS: Epithelial-conditioned media induced IEL chemotaxis, and this activity was markedly enhanced by prestimulation of epithelia with interferon-(IFN)-gamma. This chemotaxis (stimulation +) was significantly inhibited by neutralizing antibodies to IFN-gamma inducible protein-10 (IP-10) or to monokine induced by IFN-gamma (MIG). Furthermore, while high amounts of IP-10 and MIG were detected in epithelial-conditioned media after IFN-gamma stimulation, equivalent concentrations of recombinant IP-10 and MIG reproduced IEL chemotaxis. Production of IP-10 and MIG in fresh epithelial cells was supported by in situ hybridization and enzyme-linked immunosorbent assay. Lastly, fresh human IEL constitutively expressed CXCR-3 (the common receptor for IP-10 and MIG), and fresh IEL also exhibited chemotaxis to by rIP-10, rMIG, and epithelial-conditioned media. CONCLUSIONS: Epithelial cells produce chemoattractants for IEL, and such chemokine production is regulated by proinflammatory cytokines such as IFN-gamma. IP-10 and MIG may serve as potentially important epithelial chemokines for IEL, especially under inflammatory conditions.

Extrahepatic synthesis of FVII in human atheroma and smooth muscle cells in vitro.

The present series of experiments provide evidence that FVII is synthesized outside of the liver and is found in a variety of cells in normal and atherosclerotic vessels. In normal vessels FVII was localized to the endothelial cell layer and in adventitial fibroblasts at sites where tissue factor (TF) is also found. In early and advanced atherosclerotic lesions, FVII was mostly found in macrophage- rich regions colocalized with TF. Foam cells and macrophages in the necrotic core adjacent to the cholesterol clefts and foamy macrophages in early intimal thickenings all showed strong cytoplasmic staining with FVII antibodies. Although it is possible that FVII protein staining found in normal and atherosclerotic vessels originated from the blood, the finding of FVII mRNA by both in situ hybridization and RT-PCR suggests that these tissues are sites of FVII synthesis. Additional work demonstrated synthesis of FVII in a variety of tissues and smooth muscle cells and fibroblasts in vitro. The distribution of FVII synthesis in extrahepatic tissues and more recent data regarding thrombin-independent signaling as a consequence of FVII/TF binding may suggest the possibility of other cellular functions for this coagulation factor.

Perivascular responses after angioplasty which may contribute to postangioplasty restenosis: a role for circulating myofibroblast precursors?

These studies suggest that the adventitia may play a role in vascular lesion formation after balloon overstretch injury of pig coronary arteries by contributing to the cellular mass of the neointima and the synthesis of growth factors. In addition, the adventitia may contribute to vascular remodeling and constriction of the external elastic lamina through accumulation of myofibroblasts containing alpha smooth muscle actin in the adventitia surrounding the injury site. Inhibition of myofibroblast proliferation and/or recruitment by intravascular brachytherapy positively affects vascular remodeling through its action on adventitial cells. Inflammation is a major event associated with balloon angioplasty, resulting in the sequential recruitment of neutrophils (2-24 hours) and monocyte/macrophages (24-72 hours) predominantly into the adventitia surrounding the injury site. It is hypothesized that inflammatory cells release cytokines and/or increase the production of superoxides which stimulate the proliferation and recruitment of adventitial myofibroblasts. Inflammatory and proliferative responses were not confined to the local adventitia but were found extending as far as 1-3 mm away from the injured vessel in the distal perivascular tissues. Studies were performed to examine the expression of genes associated with cell migration at early times after injury in an attempt to determine the source of the adventitial myofibroblasts. Expression of genes involved in cell migration including MMP-2, MMP-9, and tenascin was found as early as 2 hours following angioplasty in the intramyocardial, pericardial, and adipose tissue fibroblasts. While these studies suggest that local tissue was the source of the myofibroblasts recruited to the injury site, we have been unable to confirm this finding by direct fluorescent labeling of adventitial cells. Recent work from our laboratory suggests that myofibroblast precursors may be isolated from buffy coat preparations from peripheral blood. These results lead us to hypothesize that stem cells that differentiate into myofibroblasts may be recruited in early inflammatory infiltrates in the adventitia. Clearly, additional work will have to be directed at a more detailed examination of the response of adventitial and other perivascular cells and tissues to balloon injury to determine their sources and their role in regulating vascular lesion development.

Regulated expression of the BTEB2 transcription factor in vascular smooth muscle cells: analysis of developmental and pathological expression profiles shows implications as a predictive factor for restenosis.

BACKGROUND: We have previously shown BTEB2, a Krüppel-like zinc finger transcription factor, to regulate expression of the SMemb/NMHC-B gene, which has been implicated in phenotypic modulation of smooth muscle cells (SMCs). The present study was done to assess the developmental and pathological expression profiles of BTEB2 and to further evaluate the clinical relevance of BTEB2 expression in human coronary artery disease. METHODS AND RESULTS: Immunohistochemistry showed developmentally regulated expression of BTEB2 with abundant expression in fetal but not in adult aortic SMCs of humans and rabbits. In balloon-injured aortas, predominant expression of BTEB2 was seen in neointimal SMCs. Atherectomy specimens obtained from primary and restenotic lesions showed predominant expression of BTEB2 to stellate SMCs. The incidence of restenosis in primary lesions was significantly higher in lesions containing BTEB2-positive cells than in lesions without (55.6% versus 25.0%, P:=0.01). CONCLUSIONS: The present study shows that BTEB2 expression is developmentally and pathologically regulated. BTEB2 is preferentially expressed in dedifferentiated or activated SMCs. Examination of human coronary artery specimens suggests that primary lesions containing BTEB2-positive cells are associated with higher risk of restenosis than BTEB2-negative lesions. These results suggest that BTEB2 can serve as a molecular marker for phenotypic modulation of vascular SMCs.

CC chemokine receptor 2 is required for macrophage infiltration and vascular hypertrophy in angiotensin II-induced hypertension.

Recent studies have identified the presence of macrophages in the arterial wall of hypertensive animals and suggested that as is the case in atherosclerosis, macrophage products may be important mediators of the adaptive response of the arterial wall. In support of this, we have previously shown that the expression of monocyte chemoattractant protein-1 is upregulated in the arteries of hypertensive animals. We hypothesized that macrophage recruitment is a critical step in the pathogenesis of hypertension. To obtain insights into this potential mechanism, we made use of mice deficient in the CC chemokine receptor 2 (CCR2), the receptor for monocyte chemoattractant protein-1. Hypertension was induced with the subcutaneous administration of angiotensin II (0.75 mg. kg(-1). d(-1)) for 7 days. Using in situ hybridization with a probe for c-fms to identify macrophages, we found that hypertension-induced macrophage infiltration of the arterial wall was virtually eliminated in CCR2-deficient mice. In addition, vascular hypertrophy was reduced by approximately 65% compared with wild-type animals. These data demonstrate that CCR2 is essential for the recruitment of macrophages into the arterial wall in the setting of hypertension. Furthermore, the decreased hypertrophic response suggests that vascular hypertrophy occurs in part as a consequence of macrophage infiltration. In angiotensin II-induced hypertension, CCR2-mediated responses are critical to the process of macrophage recruitment and vascular hypertrophy and may represent one mechanism by which at least some forms of hypertension may lead to the development of atherosclerosis.

Reperfusion induces myocardial apoptotic cell death.

OBJECTIVE: The purpose of the present study was to investigate whether apoptosis is triggered during ischemia (I) and reperfusion (R) and whether I/R-induced apoptosis is correlated with changes in expression of Bcl-2 and Bax. METHODS: Anesthetized open-chest dogs were divided into two groups. Group I: 7 h of permanent I without R (PI, n = 7); Group II: 60 min I followed by 6 h R (I/R, n = 8). Apoptosis was identified as "DNA ladder" by agarose gel electrophoresis or confirmed histologically using the terminal transferase UTP nick end labeling (TUNEL) assay. RESULTS: Collateral myocardial coronary blood flow during I, confirmed by colored microspheres was comparable in both groups. Although PI caused 72 +/- 5% infarct size, very few TUNEL-positive cells were detected in the necrotic area (0.2 +/- 0.1% of total normal nuclei), consistent with an absence of DNA laddering. In contrast, the appearance of TUNEL-positive cells was significantly displayed after 6 h R in the necrotic area in I/R group (26 +/- 4%, P < 0.001 vs. PI group), and DNA ladder occurred in all experimental animals, suggesting that myocardial apoptosis is primarily elicited by R. Densitometrically, Western blot analysis showed significant reduction in expression of Bcl-2 (16 +/- 1%) and increase in Bax (29 +/- 8%) after 6 h R in the necrotic area compared with normal tissue while expression of these two proteins was not changed in the PI group. Polymorphonuclear neutrophil (PMN) accumulation in the necrotic area determined either by immunohistochemistry with anti-CD18 antibody or by myeloperoxidase activity was significantly increased in the I/R group compared to the PI group (358 +/- 24 vs. 24 +/- 2, mm2 myocardium, P < 0.01) and (2.9 +/- 0.3 vs. 0.4 +/- 0.1, U/100 mg tissue, P < 0.01). There was a significant linear relationship between CD18-positive PMNs and TUNEL-positive cells (P < 0.05) in the I/R group. CONCLUSIONS: These results indicate that (1) PI without R did not induce apoptotic cell death, while two types of cell death, necrosis and apoptosis were found after I/R, (2) the Bcl-2 family may participate in early R-induced myocardial apoptosis, (3) PMN accumulation may play a role in the development of apoptosis.

Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart.

OBJECTIVE: Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study tested the hypothesis that IPC reduces ischemia/reperfusion-induced myocardial apoptosis by inhibiting neutrophil (PMN) accumulation and altering expression of Bcl-2 and Bax proteins. METHODS: Eighteen rats were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion with IPC (5 min ischemia and 10 min of reperfusion, n = 10) or without IPC (n = 8). Myocardial apoptosis was detected histologically using the terminal transferase UTP nick end labeling (TUNEL) assay and confirmed by DNA ladder on agarose gel electrophoresis. PMN accumulation was detected immunohistochemically with anti-rat CD18 antibody (WT3) and expression of Bcl-2 and Bax proteins was analyzed using Western blot assay. RESULTS: IPC significantly decreased TUNEL positive cells (% total nuclei) in the ischemic zone from 28.6 +/- 2.8 to 3.4 +/- 0.9 (P < 0.05), consistent with the absence of DNA ladders in the IPC group. IPC significantly attenuated PMN accumulation (cells/mm2 myocardium) in the ischemic zone from 243 +/- 19 to 118 +/- 19 (P < 0.05). By regression analysis, there was a significant correlation between TUNEL positive cells and accumulated CD18 positive PMNs in the ischemic zone (r = 0.8, P < 0.001), which was shifted downward by IPC. Densitometrically, IPC significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein in the ischemic zone from 204 +/- 57% in the control group to 76 +/- 7% (P < 0.05), while the expression of Bcl-2 was not different from the non-ischemic zone in either group. CONCLUSION: These data suggest that ischemic preconditioning may reduce myocardial apoptosis by inhibiting PMN accumulation and down-regulating expression of Bax.

Administration of adenosine during reperfusion reduces injury of vascular endothelium and death of myocytes.

INTRODUCTION: To test the hypothesis that administration of adenosine during reperfusion attenuates endothelial dysfunction and extension of infarct size by inhibiting polymorphonuclear neutrophil (PMN)-mediated events and apoptosis. METHODS: Anesthetized dogs were subjected to 1 h coronary artery occlusion and 6 h of reperfusion with infusion of saline (vehicle, n = 8) or 140 micrograms/kg per min adenosine, n = 8) continuously into the left atrium starting 5 min before reperfusion and continuing for 2 h. RESULTS: There was no intergroup difference in collateral myocardial blood flow measured by using colored microspheres in the area at risk during ischemia. Infusion of adenosine transiently improved segmental shortening (4.1 +/- 3.1% versus -2.5 +/- 2.3%, P < 0.05) and segmental work (41.4 +/- 22 versus 15 +/- 13 mmHg/mm, P < 0.05) after 4 h of reperfusion. Infusion of adenosine reduced size of infarct (determined by staining with triphenyltetrazolium chloride) from 27 +/- 2% with vehicle to 14 +/- 1%, (P < 0.05). This was confirmed by measuring that it lowered activity of plasma creatine kinase (from 19 +/- 2 versus 8 +/- 1 IU/g protein, P < 0.05). It also reduced the proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei in the perinecrotic zone from 17.3 +/- 1.6 to 10.3 +/- 1.0% (P < 0.05) and reduced the appearance of DNA ladders in gel electrophoresis. In addition, it significantly decreased accumulation of PMN in the ischemic area (determined by immunohistochemistry with anti-CD18 antibody) and activity of cardiac myeloperoxidase compared with vehicle (439 +/- 52 versus 183 +/- 20 PMN/mm2 myocardium and 1.1 +/- 0.1 versus 2.4 +/- 0.2 U/100 mg tissue, P < 0.05, respectively). Furthermore, infusion of adenosine during reperfusion preserved vascular endothelial function expressed in terms of a decrease in adherence of PMN to postischemic coronary artery endothelium (63 +/- 3 versus 36 +/- 4 PMN/mm2 endothelium, P < 0.05, basal function) and agonist (acetylcholine)-induced endothelium-dependent relaxation (negative logarithm to base 10 of concentration (mol/l) for half-maximal effect 7.7 +/- 0.1 versus 7.2 +/- 0.1, P < 0.05, stimulated function). Infusion of adenosine directly inhibited generation of superoxide radical from canine PMN in vitro dose dependently from 27.8 +/- 6.3 to 5.8 +/- 2.1 nmol/l/5 x 10(6) PMN (P < 0.05). CONCLUSION: Intra-atrial infusion of adenosine during reperfusion reduced accumulation of PMN in area at risk, preserved vascular endothelial function after ischemia-reperfusion by inhibiting interaction between PMN and endothelial cells, and decreased extension of infarct, possibly by limiting apoptosis.

Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene.

Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.

RNA diversity has profound effects on the translation of neuronal nitric oxide synthase.

A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) mRNA species revealed NOS1 to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5'-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. Elaborate splicing patterns that involve alternatively spliced leader exons and exon skipping have been superimposed on this diversity. Highly structured nNOS mRNA 5'-untranslated regions, which have profound effects on translation both in vitro and in cells, contain cis RNA elements that modulate translational efficiency in response to changes in cellular phenotype.

Ischemic preconditioning reduces neutrophil accumulation and myocardial apoptosis.

BACKGROUND: This study tested the hypothesis that ischemic preconditioning (IP) inhibits myocardial apoptosis after a short period of ischemia and reperfusion. METHODS: In 9 anesthetized dogs, the left anterior descending (LAD) coronary artery was occluded for 30 min and reperfused for 3 h (control), while in 9 others, LAD occlusion was preceded by 5 min of occlusion and 5 min of reperfusion (IP). DNA from frozen myocardial tissue samples was extracted, and apoptosis were identified as "ladders" by agarose gel electrophoresis or confirmed histologically using the terminal transferase UTP nick end-labeling (TUNEL) assay. Neutrophil accumulation was detected by measuring cardiac myeloperoxidase activity. RESULTS: Thirty minutes of LAD occlusion caused a significant decrease in blood flow (colored microspheres), which was comparable between groups. In the control group, DNA ladders occurred in the area at risk (AAR) in six out nine experiments. In contrast, DNA laddering in the AAR was not observed in any of the IP group. AAR in the control group showed a greater percentage of apoptotic cells than IP (6.7 +/- 0.9% vs 1.2 +/- 0.2%; p < 0.01). Cardiac myeloperoxidase activity (U/g tissue) was significantly reduced from 0.07 +/- 0.004 in control to 0.04 +/- 0.01 in IP group (p < 0.05). CONCLUSIONS: We conclude that ischemic preconditioning attenuates apoptosis and neutrophil accumulation in the AAR in a model of nonlethal acute ischemia and reperfusion.

The role of alpha and beta platelet-derived growth factor receptor in the vascular response to injury in nonhuman primates.

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.

Superoxide production, risk factors, and endothelium-dependent relaxations in human internal mammary arteries.

BACKGROUND: In a variety of disease states, endothelium-dependent vasodilation is abnormal. Reduced nitric oxide (NO) production, increased destruction of NO by superoxide, diminished cellular levels of L-arginine or tetrahydrobiopterin, and alterations in membrane signaling have been implicated. We examined these potential mechanisms in human vessels. METHODS AND RESULTS: Relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin, as well as superoxide production and NO synthase expression, were examined in vascular segments from patients with identified cardiovascular risk factors. Endothelium-dependent relaxations were also studied after incubation with L-arginine, L-sepiapterin, and liposome-entrapped superoxide dismutase (SOD) and after organoid culture with cis-vaccenic acid. Relaxations to acetylcholine and to a lesser extent the calcium ionophore A23187 were highly variable and correlated with the number of risk factors present among the subjects studied. Treatment of vessels with L-arginine, L-sepiapterin, liposome-entrapped SOD, or cis-vaccenic acid did not augment endothelium-dependent relaxations. Hypercholesterolemia was the only risk factor associated with high levels of superoxide; however, there was no correlation between superoxide production and the response to either endothelium-dependent vasodilator used. CONCLUSIONS: In human internal mammary arteries, depressed endothelium-dependent relaxations could not be attributed to increases in vascular superoxide production, deficiencies in either L-arginine or tetrahydrobiopterin, or reduced membrane fluidity. Variability in signaling mechanisms may contribute to the differences in responses to acetylcholine and the calcium ionophore A23187.