Perivascular graft heparin delivery using biodegradable polymer wraps.

Heparin remains the gold-standard inhibitor of the processes involved in the vascular response to injury. Though this compound has profound and wide-reaching effects on vascular cells in culture and animal models, its clinical utility has been questionable at best. It is clear that the mode of heparin delivery is critical to its potential and it may well be that routine forms of administration are insufficient to observe benefit given the heparin's short half-life and complex pharmacokinetics. When ingested orally, heparin is degraded to inactive oligomer fragments while systemic administration is complicated by the need for continuous infusion and the potential for uncontrolled hemorrhage. Thus alternative heparin delivery systems have been proposed to maximize regional effects while limiting systemic toxicity. Yet, as heparin is such a potent antithrombotic compound and since existing local delivery systems lack the ability to precisely regulate release kinetics, even site-specific therapy is prone to bleeding. We now describe the design and development of a novel biodegradable system for the perivascular delivery of heparin to the blood vessel wall with well-defined release kinetics. This system consists of heparin-encapsulated poly(DL lactide-co-glycolide) (pLGA) microspheres sequestered in an alginate gel. Controlled release of heparin from this heterogeneous system could be obtained over a period of 25 days in vitro. The experimental variables affecting heparin release from these matrices were investigated. Gel permeation chromatography (GPC) and scanning electron microscopy (SEM) were used to monitor the degradation process and found to correlate well with the release kinetics. Heparin-releasing gels inhibited growth of bovine vascular smooth muscle cells in tissue culture in a dose-dependent manner. Moreover, gel release controlled vascular injury in denuding and interposition vascular graft animal models of disease even when uncontrolled bleeding was evident with standard matrix-type release. This system may therefore provide an effective means of examining the effects of various compounds in the control of smooth muscle cell proliferation in accelerated arteriopathies and also shed light on the biologic nature of these processes.

Activation of protein kinase C inhibits the expression of connective tissue growth factor.

Connective tissue growth factor (CTGF) is a member of a protein family in which 38 cysteine residues are conserved. Although a wide variety of important biological functions have been ascribed to these proteins in recent years, the regulation of their gene expression for most members is virtually unknown. We studied the effects of protein kinase C (PKC) and tyrosine kinase on the expression of CTGF and observed that at the mRNA level CTGF expression is inhibited by the activation of PKC, but stimulated by the inhibition of PKC and tyrosine kinase. We further determined that the novel and the classical PKC isoforms are needed for the inhibition, but the atypical isoforms are not involved. Our data suggest that phosphorylation on serine/threonine and tyrosine by PKC and by tyrosine kinase are all inhibitory to the expression of CTGF.

Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro.

Connective tissue growth factor (CTGF) was first identified as a 38-kDa cysteine-rich protein which can be specifically induced by TGF-beta and was recently found to be expressed abundantly in atherosclerotic lesions, but only marginally in normal vascular tissues. It was hypothesized that CTGF is one of the factors involved in the development of atherosclerotic lesions. In this study, we investigated the functions of CTGF protein in regulating the growth and migration of vascular smooth muscle cells (VSMC) and found that by overexpressing CTGF in VSMC, proliferation and migration rates were significantly increased. The accelerated growth and migration can be reversed by an anti-CTGF antibody. In addition, overexpression of CTGF also promotes VSMC to express more extracellular matrix protein collagen I and fibronectin. Our results indicate that CTGF is a growth factor for VSMC and it may play a similar role in promoting VSMC proliferation, migration, and formation of extracellular matrix, in vivo.

Medial smooth muscle cell loss in arterial allografts occurs by cytolytic cell induced apoptosis.

OBJECTIVE: Experimental arterial allografts, used as models of chronic rejection, undergo marked loss of smooth muscle cells (SMC) from their media prior to the development of occlusive, intimal proliferative lesions. Medial SMC loss has been described in human heart transplants, and may be related to the development of occlusive coronary lesions which are the hallmark of chronic rejection. This SMC loss does not exhibit the characteristics of necrotic cell death. We sought to determine whether medial SMC loss in arterial allografts occurs by apoptosis. We further investigated these allografts for cytolytic cell-derived inducers of apoptosis, Finally, we compared two different strain combinations to assess the impact of varying histoincompatability on medial SMC loss. METHODS: Evidence for internucleosomal DNA degradation, which is characteristic of apoptosis, was sought by the in situ terminal deoxynucleotidyl transferase nick end labelling (TUNEL) method carried out on Lewis to Fisher rat femoral artery transplants (disparate at minor loci only) and Brown Norway to Lewis aortic transplants (fully disparate at major and minor loci). Isografts (Lewis to Lewis) served as controls. In a separate series of experiments graft mRNA was extracted and analysed by reverse transcription-polymerase chain reaction (RT-PCR) with primers for molecular inducers of apoptosis (TNF-alpha, Fas ligand, perforin, and granzyme-B) which are derived from cytolytic cells known to be present in allografts. RESULTS: Allograft media contained large numbers of TUNEL stained nuclei in both strain combinations. Neither isografts nor ungrafted femoral artery segments stained positive for apoptosis. RT-PCR on whole allografts in both strain combinations revealed sustained upregulation of perforin, granzyme-B, Fas-ligand and TNF-alpha mRNA concomitant with medial SMC loss. Autografts demonstrated sustained up regulation of TNF-alpha, and perforin, but only brief upregulation of granzyme-B, and no upregulation of Fas-ligand. CONCLUSIONS: These data strongly suggest that medial SMC loss in allograft arteriopathy occurs by apoptosis. Further, RT-PCR data indicate that cytolytic cell-derived inducers of apoptosis are upregulated in these grafts and may be accountable for medial SMC apoptotic cell death. Finally, fully-disparate (Brown Norway to Lewis) and minor-only incompatible (Lewis to Fisher) strain combinations both resulted in marked intimal proliferation, medial SMC loss by apoptosis, and similar patterns of expression of cytolytic cell derived inducers of apoptosis. Insofar as intimal proliferative lesion-formation may be dependent on medial damage (as in arterial-injury models), understanding the mechanism of medial SMC loss may provide a novel therapeutic approach to human cardiac transplant arteriopathy.

Involvement of hydrogen peroxide in the differentiation of clonal HD-11EM cells into osteoclast-like cells.

The present study uses the osteoclast precursor clonal line, HD-11EM, to study the potential of hydrogen peroxide (H2O2) in mediating the differentiation of HD-11EM into osteoclast-like cells. HD-11EM cells are a newly established clonal cell line that, in response to 1alpha,25-(OH)2D3, differentiate into osteoclast-like cells that are multinucleated (more than three nuclei), express tartrate-resistant acid phosphatase (TRAP), and excavate resorption pits when cultured on dentin slices in the presence of osteoblasts (Hsia et al., 1995, J. Bone Miner. Res., 10(Suppl 1):S424; Hsia, and Hauschka, 1997, unpublished data). Here we demonstrate that HD-11EM express the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase specific cytochrome b558 subunits, and that stimulation of HD-11EM with 1 or 10 nM 1alpha,25-(OH)2D3 increases the extracellular release of H2O2 within 5-10 min. Ours is the first report that stimulation of a cell with 1alpha,25-(OH)2D3 enhances the activation of NADPH-oxidase and increases the basal release of superoxide and the formation of its dismutation product, H2O2. To determine the possible involvement of H2O2 in the differentiation of HD-11EM, these cells were exposed to glucose/glucose oxidase. This enzyme system was used to deliver a pure and continuous source of H2O2 in nanomole amounts consistent with quantities produced by HD-11EM in response to 1alpha,25-(OH)2D3. Both 1alpha,25-(OH)2D3 and the exogenously generated H2O2 stimulated a dose- and time-dependent increase in TRAP activity/cell and the number of multinucleated cells 24-48 hr after treatment. Northern analysis confirmed an increase in expression of TRAP mRNA in response to either 1alpha,25-(OH)2D3 or H2O2. Decreases in cell proliferation and v-myc mRNA were also observed in response to these agents. Taken together, our findings indicate that production of H2O2 by HD-11EM is an important local factor involved in differentiation of HD-11EM into osteoclast-like cells, and suggest that H2O2 may play a role in native osteoclast differentiation.

Platelet-derived growth factor-BB, insulin-like growth factor-I, and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration.

Vascular smooth muscle cell (VSMC) migration is an important process in the development of vascular occlusive disease. To investigate mitogen regulation of VSMC migration, a cell-layer-scrape assay was used to measure migration 20 h after stimulation of VSMC with platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor I (IGF-I), or phorbol 12-myristate 13-acetate (PMA). The contributions of cell proliferation were eliminated by treatment of VSMC with hydroxyurea, which suppressed DNA synthesis.PDGF-BB stimulated VSMC migration 2.5-fold, while PMA and IGF-I stimulated migration 1.7- and 1.5-fold, respectively. The importance of protein kinase C (PKC), ERK, and phosphoinositide-3' kinase (PI3 kinase) in mitogen-stimulated migration was investigated, using specific inhibitors of these signaling molecules. PDGF-BB-stimulated migration was inhibited by the general PKC inhibitor RO 31-8220 (40%), the MEK inhibitor PD98059 (31%), and the PI3 kinase inhibitor wortmannin (22%) but not by PMA-induced downregulation of conventional and novel PKC isoforms. IGF-I-stimulated migration was inhibited by RO 31-8220 (34%) and wortmannin (37%) but was much less affected by PD98059 (19%) or PKC downregulation (10%). PMA-stimulated migration was inhibited by RO 31-8220 (53%), PD98059 (50%), wortmannin (45%), and PKC downregulation (47%). Western analysis confirmed that ERK was strongly activated by PDGF-BB and PMA but not by IGF-I. To examine potential in vivo negative regulators of VSMC migration, we analyzed the ability of heparin, an analogue of heparan sulfate, and TGFbeta to attenuate mitogen-stimulated migration. Heparin but not TGFbeta inhibited VSMC migration stimulated by all three mitogens. Delayed-addition experiments showed that RO 31-8220 retained substantial inhibitory activity even if added 3 h after PMA or IGF-I stimulation and 5 h after PDGF-BB addition, suggesting that sustained PKC activation is important for migration. The MEK inhibitor retained some effectiveness for 5 h after PDGF-BB stimulation but only 1 h after PMA addition. Western analysis showed ERK activation was transient after PMA treatment but sustained for 6 h after PDGF-BB treatment. Heparin strongly inhibited migration even if added 5-7 h after mitogen stimulation, suggesting that heparin may inhibit both short- and long-term signals necessary for migration. The present studies indicate that PMA and IGF-I activate a limited number of second messengers resulting in moderate stimulation of migration; in contrast PDGF-BB stimulates multiple signaling pathways resulting in strong stimulation of migration and lessened sensitivity to inhibitory signals.

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells.

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, there data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways.

Monocyte recruitment and neointimal hyperplasia in rabbits. Coupled inhibitory effects of heparin.

Among the many effects of heparin independent of its effects on coagulation are inhibition of vascular smooth muscle cell proliferation and regulation of leukocyte-blood vessel interactions. The potential link between these effects was examined in an animal model of vascular injury rich in inflammatory cells: the placement of endovascular metal stents in rabbit iliac arteries. Monocyte adhesion stimulated by early focal thrombus was maximal after 3 days, with infiltrating monocytes and intimal cell proliferation maximal after 7 days. Tissue monocyte number dictated cell proliferation at each time point (R2 = .92, P < .0001). Heparin reduced both early monocyte adhesion as well as monocyte infiltration within the neointima 7 and 14 days after stent placement. Reductions in adherent and tissue monocytes were commensurate with reductions in intimal cell proliferation and intimal thickening. At 14 days, heparin's inhibition of mononuclear cell adhesion was correlated with its suppression of intimal thickening (R2 = .82, P < .0001). Monocytes have been hypothesized to serve as markers, initiators, and promoters of arterial occlusive diseases. Heparin's ability to inhibit mononuclear cell adhesion and penetration and reduce neointimal size and cell proliferation after vascular injury may further implicate monocytes in the pathogenesis of neointimal hyperplasia after mechanical arterial injury.

Suppressors of oxygen metabolites fail to reduce vein graft intimal hyperplasia.

OBJECTIVE: To evaluate the role of reactive oxygen metabolites in the initiation of intimal hyperplasia of vein grafts inserted into the arterial circulation. SETTING: University pathologic research laboratory. ANIMALS: Sprague-Dawley rats. INTERVENTION: Animals were randomized to receive either the xanthine oxidase inhibitor allopurinol, the iron chelating agent starch-conjugated deferoxamine, or the free-radical scavenger 21-aminosteroid U74389G. Control animals were included for each group. The epigastric vein was inserted into the right common femoral artery. Vein grafts were harvested 30 days postoperatively. The degree of intimal hyperplasia at the two anastomoses as well as at the midgraft was calculated. MAIN OUTCOME MEASURES: The vein grafts were divided into three sections designated proximal anastomosis, midgraft, and distal anastomosis. Intimal and medial areas were determined in an observer-blind fashion and expressed as intimal area-medial area ratios. RESULTS: Pretreatment of the animals with any of these agents resulted in no significant reduction in the degree of intimal hyperplasia in any treated groups compared with the control animals 30 days postoperatively. CONCLUSIONS: Arterial reconstruction often involves interposition of vein segments into the arterial circulation. These veins are subject to ischemia and reperfusion, with the potential for generation of reactive oxygen metabolites and subsequent vein graft injury, resulting in intimal hyperplasia. We hypothesized that perioperative pharmacologic intervention either to scavenge or to reduce the production of reactive oxygen metabolites would attenuate the initial vein graft injury and thus limit the subsequent development of intimal hyperplasia. These data create doubt as to the influence of reactive oxygen metabolites in the initiation of intimal hyperplasia in the vein graft.

Upregulation and modulation of inducible nitric oxide synthase in rat cardiac allografts with chronic rejection and transplant arteriosclerosis.

BACKGROUND: The Lewis-F344 rat cardiac transplantation model produces cardiac allografts with chronic rejection characterized by arteriosclerotic lesions composed of macrophages and smooth muscle cells. Modulation of the inflammatory response with a diet deficient in essential fatty acids protects against the development of intimal thickening. Little is known about the components of the inflammatory response mediating this process. The cytokine-inducible isoform of nitric oxide synthase (iNOS) regulates the high-output nitric oxide pathway that confers activation properties to macrophages and regulates vasomotion, monocyte adherence, and smooth muscle cell proliferation in the vasculature. The purpose of the present study was to determine whether the iNOS pathway was upregulated during the course of chronic cardiac rejection. METHODS AND RESULTS: We studied iNOS mRNA and protein expression patterns in a series of Lewis-F344 cardiac allografts with early and late chronic rejection and after modulation of the inflammatory response (in an effort to attenuate arteriosclerosis). Relative gene transcript levels were measured with a 32P-dCTP reverse-transcriptase polymerase chain reaction assay designed to amplify iNOS mRNA. The distribution of the iNOS gene product was examined by immunocytochemistry with a polyclonal antibody against iNOS. NOS transcript levels increased significantly in cardiac allografts (days 7, 14, 28, and 75) compared with paired host hearts (exposed to the same circulation) and syngrafts (P < .003). Immunostaining localized the iNOS antigen within subpopulations of mononuclear inflammatory cells in cardiac allografts--presumably, activated macrophages. The number of iNOS-positive mononuclear cells was 25-fold higher in cardiac allografts compared with paired host hearts and syngrafts (P < .009). In cardiac allografts of 75 days or older, there also was striking iNOS staining within some medial and intimal smooth muscle cells in various vessels. Modulation of the inflammatory response (with a diet deficient in essential fatty acids) produced significant decreases in the intimal thickening score and in the percentage of diseased vessels in 28-day cardiac allografts compared with allografts from rats fed a control diet. There was a correlate decrease in iNOS transcript levels and in the number of iNOS-positive mononuclear cells in the 28-day cardiac allografts from rats fed the essential fatty acid-deficient diet. CONCLUSIONS: The early and persistent upregulation of iNOS in chronic cardiac rejection and the coincident reduction in arteriosclerosis and downregulation of iNOS suggest that this inducible regulator may contribute to the inflammatory response mediating transplant arteriosclerosis.

Intrathymic tolerance in the Lewis-to-F344 chronic cardiac allograft rejection model.

Successful induction of donor-specific unresponsiveness by intrathymic inoculation of alloantigen in several experimental acute rejection models has led us to hypothesize that similar immune manipulations can prevent chronic rejection and development of graft arteriosclerosis in the Lewis-to-F344 rat chronic cardiac allograft rejection model. Recipient F344 rats were treated with donor (Lewis) splenocytes by intrathymic injection (i.t.) alone (10 x 10(6) cells/lobe); with donor splenocytes i.t. plus a one-time dose of ALS (1 mg) by intraperitoneal injection (i.p.); or with ALS i.p. (1 mg) alone 2 and 6 weeks prior to heterotopic Lewis heart transplantation. Control F344 recipients received saline i.t. Allografts were monitored by daily palpation, and long-term surviving grafts were harvested on day 90 for histopathologic analysis. Control allografts had 28.6% long-term survival (> 90 days) with mean graft survival of 46.7 +/- 12.2 days. At day 90 the surviving control allografts were enlarged and fibrotic with barely palpable heartbeat (mean heartbeat grade 0.29 +/- 0.18), and histologically showed diffuse moderate mononuclear cell infiltrates and advanced graft arteriosclerosis (mean vessel score 3.57 +/- 0.10 and 89 +/- 1% vessels diseased). Recipient treatment with intrathymic donor splenocytes alone significantly prolonged graft survival (89% long-term survival; mean 83.8 +/- 6.2 days, P < 0.04), but did not significantly inhibit the development of graft arteriosclerosis (score 2.98 +/- 0.53 and 79 +/- 8% diseased, P = NS). By contrast, treatment with i.t. donor splenocytes plus ALS 2 weeks prior to transplantation prolonged graft survival (100% long-term; mean 90.0 +/- 0.0 days, P < 0.04), and markedly inhibited graft arteriosclerosis (score 0.80 +/- 0.14, P < 0.05; 27 +/- 4% diseased, P < 0.05). ALS alone given two weeks prior to transplantation also prolonged graft survival (100% long-term; mean 90.0 +/- 0.0 days, P < 0.04), and inhibited graft arteriosclerosis (score 0.89 +/- 0.31, P < 0.05; 25 +/- 7% diseased, P < 0.05). However, when ALS was given 6 weeks prior to heart transplantation the beneficial effect of ALS alone was abolished, suggesting that lymphocyte depletion may have been responsible for the observed effects when ALS was administered at 2 weeks. Interestingly, intrathymic donor splenocytes plus ALS 6 weeks prior to transplantation, on the other hand, showed significant prolongation of allograft survival (100% long-term, mean 90.0 +/- 0.0 days, P < 0.04), and inhibited graft arteriosclerosis (score 0.41 +/- 0.02, P < 0.05; 16 +/- 2% diseased, P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Upregulation of cytokines associated with macrophage activation in the Lewis-to-F344 rat transplantation model of chronic cardiac rejection.

Lewis-to-F344 rat cardiac allografts develop chronic rejection and arteriosclerotic lesions rich in mononuclear cells (especially macrophages). This study was performed to determine whether cytokine pathways associated with macrophage activation are upregulated in hearts undergoing chronic rejection. Gene transcript levels for IFN-gamma, monocyte chemoattractant protein-1 (MCP-1), and IL-6 were measured with reverse-transcription PCR assays optimized for each gene. Gene products were confirmed by immunohistology. For all three genes, transcript levels in rat cardiac allografts increased significantly on day 7 and remained elevated on days 14 and 28 posttransplantation, as compared with naive hearts, paired host hearts, and syngrafts (P < 0.006). For the inducible genes IFN-gamma and MCP-1, high transcript levels in cardiac allografts were in contrast with low levels in host spleens. On the other hand, transcript levels for the basally expressed gene IL-6 were elevated in both organs. Immunostaining confirmed allograft-specific expression for all three cytokines and localized the gene products to infiltrating mononuclear cells in the interstitium and vasculature. The sustained expression of these cytokines in cardiac allografts undergoing chronic rejection supports the widely held hypothesis that the intimal changes associated with transplant arteriosclerosis are mediated by cellular activation and cytokine production.

CD4+ mononuclear cells induce cytokine expression, vascular smooth muscle cell proliferation, and arterial occlusion after endothelial injury.

Studies of T cell-deficient or immunosuppressed animals undergoing arterial endothelial denudation have yielded conflicting results as to the contribution of the immune system to neointimal vascular smooth muscle cell accumulation and proliferation. We investigated the cell types and cytokine expression associated with intimal hyperplasia occurring 14 days after balloon angioplasty of the carotid artery in Sprague-Dawley rats. Immunohistological studies using monoclonal antibodies showed that the carotid luminal occlusion observed was associated with smooth muscle cell proliferation and neointimal accumulation of large numbers of CD4+, ED1+ mononuclear cells but no T cells. There was also wide-spread staining for the inflammatory cytokine interleukin-1B (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and IL-8, as well as dense expression of the potent smooth muscle mitogens platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and protein S. The relationship of smooth muscle cell proliferation to monocyte/macrophage accumulation and cytokine expression was tested by daily intraperitoneal administration for 14 days of a rat CD4-specific monoclonal antibody, BWH-4 (500 micrograms/day). Morphometric analysis at day 14 showed that the intimal area of animals treated with CD4 monoclonal antibody comprised 7% +/- 4% of the arterial wall compared with 50% +/- 6% in control animals (n = 6/group, P < 0.001). In addition, immunohistological studies showed that CD4 monoclonal antibody treatment markedly reduced the intimal accumulation of mononuclear and smooth muscle cells and essentially abrogated expression of the cytokines PDGF-BB, TGF-beta, IL-1 beta, TNF-alpha, and IL-8, plus the anticoagulant molecule, protein S. Our results document the extensive expression in vivo of cytokines that in vitro promote vascular smooth muscle cell proliferation, and suggest that CD4+ mononuclear cells or their secreted products play a key role in the pathogenesis of intimal hyperplasia after endothelial injury. Furthermore, these observations may have clinical relevance in the development of novel strategies to prevent arteriosclerosis.

NADPH-oxidase expression and in situ production of superoxide by osteoclasts actively resorbing bone.

Recent reports have suggested that production of superoxide or other reactive oxygen species by activated osteoclasts may play a role in the complex process of bone resorption; however, the enzyme responsible for production of superoxide by osteoclasts has not been characterized. To determine if osteoclasts express NADPH-oxidase, a superoxide-generating enzyme found in phagocytic leukocytes, immunohistochemical studies were performed on tibia from 1-5-d-old rats using mAbs 449 and 48 and an antiserum specific for p47-phox. These antibodies recognize epitopes on the alpha and beta subunits of cytochrome b558, respectively, and the p47 cytosolic component of NADPH-oxidase. We found that osteoclasts attached to bone surfaces in tibia expressed all three components, as did mature polymorphonuclear and some mononuclear leukocytes in the bone marrow. In many adherent osteoclasts, the cytochrome b558 subunits were localized to the ruffled-border and bone interfaces. Studies were also performed on mature rat tibia that had undergone controlled fracture. By two weeks the healing fractures develop a callus rich in actively resorbing osteoclasts. Osteoclasts within the calluses, and attached to bone surface, also expressed the cytochrome b558 proteins. In addition to demonstrating the expression of NADPH-oxidase, the active production of superoxide by osteoclasts was also demonstrated in situ in freshly isolated tibia using 3,3'-diaminobenzidine (DAB)-Mn2+, a histochemical method specific for superoxide localization. Osteoclasts attached to bone surfaces contained deposits of oxidized DAB which were observed by light microscopy. Nonstimulated polymorphonuclear and mononuclear leukocytes in the bone marrow did not contain DAB deposits unless stimulated with phorbol myristate acetate, a known activator of NADPH-oxidase. These findings indicate that osteoclasts contain NADPH-oxidase, and during the process of resorbing bone, are actively producing superoxide.

Identification and upregulation of galactose/N-acetylgalactosamine macrophage lectin in rat cardiac allografts with arteriosclerosis.

Using differential mRNA display to uncover potential mediators associated with chronic rejection, we identified a cDNA fragment induced in Lewis to F344 rat cardiac allografts with arteriosclerosis but not Lewis syngrafts. The full-length cDNA (1.4 kb) isolated from a rat cardiac allograft cDNA library was 99% identical to galactose/N-acetylgalactosamine (Gal/GalNAc) macrophage lectin, a cell-surface receptor. This cDNA hybridized in Northern analysis with total RNA from eight cardiac allografts but not with host hearts, syngrafts, or other organs. There was a significant allograft-specific increase in transcript levels measured by reverse transcriptase PCR at days 7, 14, 28, and 75 in comparison with paired F344 host hearts (subject to same circulation but histologically normal), day-0 hearts, and syngrafts (P < 0.008, n = 4 at each time). Transcript levels in cardiac allografts were higher than those in paired host spleens (a major source of inflammatory cells) (P < 0.0001), indicating the localized nature of Gal/GalNAc lectin induction. By in situ hybridization and immunostaining, Gal/GalNAc lectin expression localized to a subset of inflammatory cells in cardiac allografts. These findings link Gal/GalNAc macrophage lectin to the chronic rejection process, as a possible mediator of macrophage infiltration.

Robert Feulgen Lecture 1994. Cytochemistry and reactive oxygen species: a retrospective.

This retrospective reviews the methodology we have developed over several decades for detecting reactive oxygen species (ROS), using the activated polymorphonuclear leukocyte (PMN) as the paradigm of a cell which vigorously generates ROS through activation of NADPH oxidase. In the seventies, the sites of ROS generation by PMN were not clear from biochemical data, and we sought to develop new methods for the cytochemical localization of O2.-, H2O2, and the H2O2-myeloperoxidase (MPO)-halide system. The H2O2-MPO-halide system in phagocytosing cells was localized at the fine structural level by our development of 3,3'-diaminobenzidine (DAB) as a cytochemical probe for detecting peroxidase activities. Using DAB and exogenous H2O2, we confirmed that azurophil granules discharged MPO into the phagosome, and using particles coated with DAB and relying on endogenous H2O2 to yield oxidized DAB, H2O2 was localized to phagolysosomes. The subcellular sites of H2O2 generation were shown using cerium ions which react with H2O2 and precipitate electron opaque cerium perhydroxides (Ce(OH)2OOH and Ce(OH)3OOH). The results suggested that NADPH oxidase is associated with the plasma lemma, and that the enzyme enters the phagosome along with the invaginating plasmalemma, accounting for the presence of H2O2 in the phagosome. As O2.- is the major product of NADPH oxidase, its detection was of some importance. Based on the concept that O2.- oxidizes Mn2+ to Mn3+, and Mn3+ oxidizes DAB, a medium containing DAB-Mn2+ was used to localize sites of O2.- production in stimulated PMN. The localizations were, as expected, similar to those for H2O2. These techniques have been of considerable usefulness and in general provide the foundation for cytochemistry of ROS in other systems.

Chronic cardiac rejection: identification of five upregulated genes in transplanted hearts by differential mRNA display.

Transplant arteriosclerosis, the major manifestation of chronic rejection, develops after allogeneic (Lewis to F344) but not syngeneic (Lewis to Lewis) rat cardiac transplantation. To identify transcriptionally regulated mediators associated with chronic cardiac rejection, we adapted the differential mRNA display technique for in vivo transplant specimens. Gene transcript patterns in four allogeneic hearts showing early signs of chronic rejection were compared with those in two syngeneic hearts exposed to the same surgical procedure but histologically normal. Twelve differentially expressed cDNA bands were identified. We improved the probability of isolating one or more allograft-specific cDNAs from a single display band by first using recovered and reamplified PCR products as probes in RNA blot analysis. cDNA fragments cloned from individual bands were then used in a second RNA blot analysis, which allowed for the correlation of specific mRNA transcripts with cDNA clones. Five cDNA clones produced time-dependent, allograft-specific hybridization. Sequence analysis demonstrated that two of these cDNAs corresponded to unknown genes, whereas the other three represented known genes not previously associated with chronic rejection. The latter group included the macrophage lectin specific for galactose/N-acetylgalactosamine (a cell-surface receptor), the nuclear P1 gene (a homologue of a yeast replication protein), and a ubiquitin-like gene. Our application of the differential display technique allowed the direct identification of potential mediators under in vivo conditions that preserve the environment of the disease process--including infiltrating cell populations critical to the inflammatory response.