Microsecond-pulsed dielectric barrier discharge plasma stimulation of tissue macrophages for treatment of peripheral vascular disease.

Angiogenesis is the formation of new blood vessels from pre-existing vessels and normally occurs during the process of inflammatory reactions, wound healing, tissue repair, and restoration of blood flow after injury or insult. Stimulation of angiogenesis is a promising and an important step in the treatment of peripheral artery disease. Reactive oxygen species have been shown to be involved in stimulation of this process. For this reason, we have developed and validated a non-equilibrium atmospheric temperature and pressure short-pulsed dielectric barrier discharge plasma system, which can non-destructively generate reactive oxygen species and other active species at the surface of the tissue being treated. We show that this plasma treatment stimulates the production of vascular endothelial growth factor, matrix metalloproteinase-9, and CXCL 1 that in turn induces angiogenesis in mouse aortic rings in vitro. This effect may be mediated by the direct effect of plasma generated reactive oxygen species on tissue.

Overexpression of allograft inflammatory factor-1 promotes proliferation of vascular smooth muscle cells by cell cycle deregulation.

Allograft inflammatory factor-1 (AIF-1) is not present in normal arteries, but its expression is induced in vascular smooth muscle cells (VSMCs) in several models of arterial injury. The proliferation of VSMCs is a major component of neointimal hyperplasia in many arteriopathies, and the purpose of this study was to determine the role of AIF-1 in the growth of VSMCs. Transfection and constitutive expression of AIF-1 in a primary and a rat VSMC line results in enhanced growth of those cells as measured by cell number and is proportional to the amount of AIF-1 expressed. Constitutive expression of AIF-1 results in a shorter cell cycle, as measured by flow cytometry, and aberrant expression of cell cycle proteins, as determined by Western blot. AIF-1 overexpression also permits growth of these cells in serum-reduced media. Collectively, these data suggest that AIF-1 may participate in the progression of vascular proliferative disease on the basis of its ability to regulate the growth of VSMCs.

Expression of anaphase-promoting complex 5 in balloon angioplasty-injured rat carotid arteries and mitogen-stimulated human vascular smooth muscle cells.

We utilized differential display to identify differentially expressed mRNAs induced by balloon angioplasty injury. A recently described subunit of the anaphase-promoting complex, APC5, is not expressed in uninjured rat carotid arteries. Expression of APC5 mRNA is detectable as early as 1 day post-injury, reached maximal levels by 3-7 days, and declined by 14 days post-injury. APC5 is not expressed in serum-starved cultured human vascular smooth muscle cells (VSMC), but is strongly induced by mitogenic factors, and to a lesser extent by nonmitogenic cytokines. The kinetics of APC5 expression is associated with cell cycle progression, and corresponds with PCNA expression in PDGF-stimulated human VSMC. Interestingly, APC5 is expressed in most human tissues examined, regardless of their proliferative state. These data are the first description of the cytokine-inducible expression of APC5 and suggest that expression of this gene may represent an important event in the pathogenesis of vascular proliferative diseases.

Expression of allograft inflammatory factor-1 is a marker of activated human vascular smooth muscle cells and arterial injury.

The cytokine-induced activation and proliferation of medial vascular smooth muscle cells (VSMCs) leading to intimal hyperplasia is one of the most critical cellular events in the formation of transplant arteriopathy and balloon angioplasty-induced restenosis. Allograft inflammatory factor-1 (AIF-1) is a calcium-binding protein that we have previously shown to be expressed in balloon angioplasty-injured rat carotid arteries. We hypothesized that AIF-1 expression may be associated with the VSMC response to injury. In this study, we examined AIF-1 expression in immunologic and mechanical models of arterial injury. Reverse transcription-polymerase chain reaction and Western analysis demonstrated that AIF-1 is acutely and transiently expressed in aortic medial smooth muscle cells of rat cardiac allografts, with mRNA and protein peaking at 3 to 7 days after transplant and declining by 10 days after transplant. Immunohistochemical analysis identified abundant AIF-1 in the medial VSMCs of these vessels. Immunohistochemical analysis of balloon angioplasty-injured swine coronary arteries also demonstrates an acute AIF-1 expression detectable by 24 hours and continuing up to 14 days after the procedure. AIF-1 in these vessels also localizes to the medial VSMCs and cells of the developing neointima. AIF-1 protein is not expressed in quiescent cultured human VSMCs but is induced in cells challenged with various inflammatory cytokines, primarily by interferon-gamma, interleukin-1beta, and T-cell-conditioned media. Transfection and overexpression of AIF-1 in human VSMCs result in enhanced growth of these cells. Taken together, these data indicate that AIF-1 expression is associated with vascular trauma and suggest that this protein may play a role in VSMC activation subsequent to arterial injury.

14-3-3Gamma interacts with and is phosphorylated by multiple protein kinase C isoforms in PDGF-stimulated human vascular smooth muscle cells.

It has recently been demonstrated that some members of the 14-3-3 protein family play an important role in signal transduction leading to cellular proliferation. We have previously shown that expression of 14-3-3gamma is induced by growth factors in human vascular smooth muscle cells (VSMC). In this study, we cloned the human homolog of 14-3-3gamma and observed many potential phosphorylation sites, suggesting the potential for post-translational modification. In VSMC treated with platelet-derived growth factor (PDGF), 14-3-3gamma protein was expressed and phosphorylated in an activation-dependent manner. Platelet-derived growth factor-induced phosphorylation could be inhibited by phosphokinase C (PKC) inhibitory compounds, and 14-3-3gamma could be phosphorylated in the absence of PDGF by compounds that activate PKC. We also demonstrated interaction between 14-3-3gamma and several PKC isoforms (alpha, beta, gamma, theta, and delta), implicating these PKC family isoforms as the kinases responsible for PDGF-induced 14-3-3gamma phosphorylation. We found that 14-3-3gamma interacted with the signal transduction protein Raf-1, suggesting that 14-3-3gamma provides a link between this protein and PKC. Thus, 14-3-3gamma may represent a signal transduction protein that is regulated transcriptionally and post-transcriptionally by growth factors.

Endothelin-1 as a putative modulator of gene expression and cellular physiology in cultured human corporal smooth muscle cells.

PURPOSE: Increases in cytosolic calcium levels trigger smooth muscle contraction while nuclear calcium increases are thought to regulate gene expression. Endothelin-1 (ET-1) affects both. The goal of these studies was to further investigate the importance of ET-1 to corporal physiology by examining its actions on proliferation and immediate early gene (IEG) expression in cultured human corporal smooth muscle cells. MATERIALS & METHODS: Early passage (1-3) smooth muscle cells were grown in culture and exposed to either phenylephrine (PE) or ET-1 in the absence and presence of serum, the ET(A) or ET(B) selective antagonist BQ123 or IRL1038, or the L-type Ca2+ channel blocker, verapamil. Cell proliferation was assessed with a hemocytometer. The effects of ET-1 on c-myc and c-fos were evaluated using Northern blot analysis. Parametric or nonparametric statistics were used as appropriate. RESULTS: Addition of ET-1 (100 nM) to serum-starved cultured corporal smooth muscle cells was associated with a nearly 2-fold increase in cell number, as well as 2 to 6-fold increases in c-myc and c-fos levels. Cellular proliferation was inhibited by ET(A)- or ET(B)-receptor subtype blockade with BQ123 (1 microM) or IRL1038 (1 microM), respectively, or blockade of Ca2+ channels with verapamil (10 microM). PE (3 microM) had no detectable effect on smooth muscle proliferation. CONCLUSIONS: Cell proliferation was mediated by activation of the ET(A) and ET(B) receptor subtypes, dependent on transmembrane Ca2+ flux, and correlated with significant increases in c-myc and c-fos mRNA levels. These studies extend previous observations to indicate the potential pleotropic actions of this peptide in the regulation of human corporal smooth muscle physiology in vivo.

IRT-1, a novel interferon-gamma-responsive transcript encoding a growth-suppressing basic leucine zipper protein.

Interferon-gamma (IFN-gamma) inhibits proliferation of vascular smooth muscle cells (VSMCs) in culture and reduces arterial restenosis post-balloon angioplasty. The identification and characterization of IFN-gamma-specific transcripts in VSMCs are an important approach to discern the molecular mechanisms underlying vascular proliferative disease. In this report, we describe IRT-1, a novel mRNA transcript constitutively expressed in a number of human tissues, but expressed in human VSMCs only when they are stimulated with IFN-gamma. This mRNA expression is induced >200-fold 72 h after IFN-gamma treatment. IRT-1 mRNA is also acutely expressed in rat carotid arteries that are injured by balloon angioplasty. The IRT-1 cDNA transcript encodes a basic protein that contains a leucine zipper motif, a core nuclear localization sequence, and a single strongly hydrophobic region. Constitutive IRT-1 mRNA expression in human peripheral blood lymphocytes is reduced when these cells are stimulated to proliferate. Overexpression of IRT-1 protein in VSMCs alters their morphology and dramatically reduces their proliferative capacity. This study suggests that IRT-1 is an IFN-gamma-inducible factor that may regulate the progression of vascular proliferative diseases.

Lymphocyte-specific inducible expression of potassium channel beta subunits.

Many studies have shown that voltage-gated potassium (Kv) channel activity is essential for T-lymphocyte proliferation. The IL-2-inducible neuroimmune gene, I2rf5 is the mouse homologue of the rat Kv beta 2 subunit. In this study we show that in addition to constitutive expression in adult murine brain, expression of Kv channel subunits beta 1.1 and beta 2.1 is inducible in a cloned T-helper cell line stimulated with IL-2 and in normal murine splenocytes stimulated with Con A or LPS. This expression pattern appears to be lymphocyte specific, because stimulated fibroblasts and vascular smooth muscle cells do not express Kv beta channel subunit mRNA. These observations suggest that Kv beta subunit expression is tissue specific and inducible in stimulated lymphocytes. Because Kv beta subunits modulate K+ channel activity, the inducible and variable expression of these subunits in lymphocytes may represent an additional regulatory mechanism for lymphocyte proliferation.

cDNA cloning of human allograft inflammatory factor-1: tissue distribution, cytokine induction, and mRNA expression in injured rat carotid arteries.

We have previously identified a partial cDNA fragment obtained from rat carotid arteries subject to balloon angioplasty which is increased in response to vascular injury. We now report the isolation and characterization of a full length human clone which is 98% homologous at the amino acid level to the rat. Allograft Inflammatory Factor-1 (AIF-1), a gene previously reported to be expressed in rat IFN gamma-activated macrophages. This transcript is expressed in low levels in undamaged, and increases 1 day and 3 days, and declines 7 days post balloon angioplasty. This human AIF-1 homologue is inducible in serum and cytokine-stimulated human smooth muscle cells. Interestingly, human tissue distribution indicates a constitutive expression in lymphoid tissue which can be augmented by mitogens. In summary, human AIF represents a cytokine inducible, tissue-specific, and highly conserved transcript transiently expressed in response to vascular trauma.

Expression of 14-3-3 gamma in injured arteries and growth factor- and cytokine-stimulated human vascular smooth muscle cells.

One of the most critical cellular events in disease states such as vascular restenosis is the cytokine-induced activation of vascular smooth muscle cells (VSMCs) resulting in intimal thickening. Identification of the molecular mechanisms of VSMC activation is crucial in understanding the initiation of vascular restenosis. In this report, we show that one 14-3-3 protein family isoform, gamma, is transcriptionally up-regulated in rat carotid arteries after balloon angioplasty. 14-3-3 gamma protein induction localizes to both the media and neointima in such injured vessels. Because it has been shown that some members of the 14-3-3 family may play an important role in cellular proliferation by binding to and activating the protein kinase Raf-1 and VSMCs constitute the major cellular component of the restenotic lesion, we investigated the expression of this message in serum- and cytokine-stimulated human VSMCs. Both serum and selected cytokines induce 14-3-3 gamma mRNA and protein, the magnitude of which correlates with the degree of cellular stimulation. 14-3-3 gamma mRNA, however, does not increase when other cell types are stimulated with specific growth factors. Human tissue distribution of 14-3-3 gamma mRNA indicates that in contrast to other 14-3-3 proteins, the gamma isoform is highly expressed in VSMCs and skeletal and heart muscle, suggesting an important role for the gamma isoform in muscle tissue as well. These results indicate that 14-3-3 gamma expression increases in response to vessel damage and proliferative signals and may implicate a role for the gamma isoform of 14-3-3 in VSMC activation and metabolism.

Identification of a down-regulated mRNA transcript in corpus cavernosum from diabetic patients with erectile dysfunction.

Fifty per cent of men with diabetes have erectile dysfunction. Previous studies demonstrated that cultured smooth muscle cells from corpus cavernosum display significantly altered K+ channel function, PGE-induced cAMP accumulation, and endothelin-1 induced Ca2+ mobilization that are consistent with the pathophysiology of erectile dysfunction. Since defects in signal transduction frequently lead to altered gene expression, we examined differences in gene expression in corporal tissue excised from three diabetic patients with erectile dysfunction function and one patient with neurogenic erectile dysfunction. Using differential display, we identify a transcript expressed in tissue derived from the patient with impotence secondary to a radical prostectomy, but which was greatly reduced or absent in corporal tissue from all three diabetic patients examined. DNA sequence analysis indicates that this transcript has no significant homology to sequences presently deposited in the GenBank database. This suggests that altered gene expression may play a significant part in the etiology of erectile dysfunction.

Isolation and characterization of BART-1: A novel balloon angioplasty responsive transcript in rat carotid arteries.

In previous studies, differential display analysis of balloon angioplasty-damaged rat carotid arteries identified a temporally expressed partial cDNA termed RC9. This message is undetectable in undamaged vessels, reaches maximal levels 3 days post-procedure, and reduces to half-maximal expression by 14 days post angioplasty. Using RC9 to screen a cDNA library, we now report the isolation and characterization of a full-length clone, termed BART-1. BART-1 is 98% homologous to RC9 and shows the same mRNA expression pattern as RC9 in rat carotid arteries subject to balloon angioplasty. Northern analysis of various rat tissues reveals tissue specificity and possible differential processing. Neither the nucleic acid nor amino acid sequences demonstrate similarity to previously reported expressed sequences. Predicted amino acid analysis reveals two strongly hydrophilic and one hydrophobic region, suggesting a type II integral membrane protein. The cDNA sequence hybridized to genomic DNA from a variety of species, suggesting evolutionary conservation. Thus, BART-1 mRNA appears to represent an inducible, tissue-specific transcript encoding a putative integral membrane protein transiently expressed in response to vascular trauma.

Antisense oligonucleotides to the p65 subunit of NF-kB inhibit human vascular smooth muscle cell adherence and proliferation and prevent neointima formation in rat carotid arteries.

Neointima formation associated with vascular restenosis is a complex local inflammatory process actively involving the major cellular component of the atherosclerotic lesion, the vascular smooth muscle cell. NF-kB is a pleotrophic transactivator of a diverse group of genes whose activation has been strongly associated with the cellular response to inflammation. We treated human vascular smooth muscle cells (VSMC) with phosphorothio antisense oligonucleotides to the p65 subunit of NF-kB and report that addition of p65 antisense oligonucleotides (1-20 microM), but not sense or p50, inhibit human VSMC adherence and proliferation in a concentration-dependent manner. Additionally, administration of p65 antisense significantly inhibited neointima formation in balloon angioplasty treated rat carotid arteries, indicating that the p65 subunit of NF-kB transactivates genes whose expression is important in VSMC pathobiology. These results suggest that abrogation of p65 reduces neointima formation by inhibition of smooth muscle cell proliferation and adherence.

Use of differential display to identify differentially expressed mRNAs induced by rat carotid artery balloon angioplasty.

BACKGROUND: We have used differential display technology in an animal model of arterial restenosis to study the molecular mechanisms of neointima formation. EXPERIMENTAL DESIGN: Balloon angioplasty of the rat common carotid artery was performed, and tissues were examined from naive animals (control) and from animals that had undergone angioplasty 6 hours, 3 days, and 14 days earlier. Total RNA was isolated, and differential display was performed to identify mRNA transcripts whose expression is modulated in a temporal fashion as a consequence of balloon angioplasty. RESULTS: Using total RNA isolated from vessels excised from naive rats and those from rats that underwent angioplasty up to 14 days earlier, we describe two differentially regulated transcripts by differential display and verify the expression pattern of these same transcripts by Northern analysis. DNA sequence analysis has identified one of these angioplasty-induced transcripts as a novel sequence and the other as the tryptophan hydroxylase and protein kinase C regulatory protein 14-3-3 gamma. CONCLUSIONS: In this study, we describe the in vivo application of the differential display technique in a rat carotid artery angioplasty model to identify mRNA transcripts whose expression is modulated selectively in vessels undergoing neointima formation. Use of this technique in animal models of disease should facilitate our understanding of gene expression patterns in pathogenesis and may serve as a reliable technique to "fingerprint" disease processes.

Genomic organization and genetic mapping of the neuroimmune gene I2rf5 to mouse chromosome 4.

The nervous and immune systems share many functional and molecular similarities, including shared surface antigens, secretions of soluble factors, and cross-modulatory effects. We have identified previously a novel mRNA termed F5, which is expressed only in activated T lymphocytes and mature, postmitotic neurons. Tissue specificity and sequence conservation suggest an important function for F5 in T-lymphocyte proliferation and neuronal maturation. The F5 gene product is an evolutionarily conserved, cytoskeletal-associated phosphoprotein. A full-length mouse genomic clone has been isolated. The protein coding region of the F5 gene is approximately 16 kb in length and is composed of 13 coding exons. The gene encoding F5, termed I2rf5, was mapped using interspecies mouse crosses in close proximity to a number of genes associated with neuronal defects on distal chromosome 4.

A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.

Increased expression of the inositol 1,4,5-trisphosphate receptor in human leukaemic (HL-60) cells differentiated with retinoic acid or dimethyl sulphoxide.

The Ins(1,4,5)P3 receptor was examined in human promyelocytic leukaemic cells (HL-60) and in HL-60 cells differentiated towards granulocytes with either retinoic acid (RA) or dimethyl sulphoxide (Me2SO). HL-60 cell membranes enriched in marker enzyme activities of the endoplasmic reticulum and the plasma membrane possess a high-affinity binding site for [3H]Ins(1,4,5)P3 (KD = 22 nM). Electrotransfer studies indicate that Ins(1,4,[32P]5)P3 binds specifically to a 260 kDa protein of HL-60 cell membranes. This Ins(1,4,5)P3-binding protein selectively binds Ca(2+)-mobilizing inositol phosphates and other inositol phosphates which also bind to the purified InsP3 receptor, suggesting that the Ins(1,4,5)P3-binding protein of HL-60 cell membranes is the InsP3 receptor. When HL-60 cells are incubated with 1 microM-RA or with 1.25% Me2SO the cells differentiate within 5-7 days into cells resembling neutrophils in both structure and function. Treated cells cease to proliferate, acquire the ability to reduce Nitro Blue Tetrazolium dye, and undergo morphological changes typical of differentiated granulocytes. Concomitant with HL-60 cell differentiation, the maximal [3H]Ins(1,4,5)P3 binding in membranes increases 3-4-fold, with no change in KD. The results suggest that there is an absolute increase in the level of the InsP3 receptor during HL-60 cell differentiation and that the expression of this signal-transducing protein may be specifically regulated by differentiation factors.

Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.