Morris J. Karnovsky

Ultrastructural localization of several phosphatases with cerium

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.

Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries.

Basic fibroblast growth factor (bFGF) is mitogenic for smooth muscle cells (SMC) and angiogenic. We examined the in vivo effects of bFGF in balloon denuded carotid arteries of laboratory rats. bFGF was administered continuously from polymer-based devices at 34 ng/d into the periadventitial space of rat carotid arteries for 2 wk. Intimal hyperplasia was not observed in the absence of injury or with lipopolysaccharide induced endothelial dysfunction. Different degrees of vascular injury produced proportionally more intimal hyperplasia. bFGF increased the intimal hyperplastic response 1.3-fold with severe vascular injury, and 2.4-fold with more mild injury. Increased cell proliferation, not extracellular matrix production, accounted for these effects. Cell density was unchanged for the control and bFGF-treated groups, and the number of proliferating intimal cells at 2 wk rose to an amount equivalent to the increase in mass; 1.9- and 4.0-fold for severe and lesser injury, respectively. The relative ability of heparin to reduce SMC proliferation was not altered by the presence of bFGF.bFGF also induced profound angiogenesis within and surrounding the polymeric releasing device, and in the vasa vasorum immediately around the injured arteries. bFGFs effect on vasa was linearly related to the amount of SMC proliferation within the blood vessel. Thus, the in vivo mitogenic and angiogenic potential of bFGF are coupled, and may be similarly modulated by the products of local injury and/or factors in the vessel wall.

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus.

Experimental graft arteriosclerosis. I. The Lewis-to-F-344 allograft model.

Progressive graft arteriosclerosis is responsible for the majority of late deaths occurring in cardiac transplant recipients. In order to define a model of this disease in the rat, we exchanged heterotopic cardiac allografts between MHC-compatible inbred strains. Lewis rats served as donors and F-344 rats as recipients. Twenty allografts were followed by daily palpation and removed at the time of terminal rejection or on the 120th postoperative day for pathologic study. Sixteen allografts (80%) survived at least three weeks, and five allografts (25%) survived indefinitely. The majority of arteries (greater than 90%) examined demonstrated significant intimal disease; histologic findings in lesions in allografts rejecting at early time points included intense mononuclear cell infiltration of the intima, while lesions in long-term-surviving allografts demonstrated fibrous intimal thickening, which is characteristic of graft arteriosclerosis seen clinically. A limited course of cyclosporine therapy in F-344 recipients increased the incidence of indefinite allograft survival from 25% to 86%, and was associated with a modest reduction in the amount of intimal disease observed. These results suggest that this model should be useful in future studies regarding the pathogenesis and therapy of cardiac graft arteriosclerosis.

Inhibition of experimental neointimal hyperplasia and thrombosis depends on the type of vascular injury and the site of drug administration.

BACKGROUND Heparin inhibits vascular smooth muscle cell proliferation in tissue culture and limits neointimal hyperplasia after experimental arterial injury but has been ineffective in reducing clinical restenosis. We examined how this discrepancy might reflect suboptimal drug-tissue interactions and/or differences in the vascular response to injury. METHODS AND RESULTS Intravenous infusion was compared with local administration of heparin to injured rabbit iliac arteries either from drug-impregnated polymeric controlled release matrices in the perivascular space or from drug-releasing endovascular stents. Occlusive thrombosis, seen in 42% of control stent-bearing arteries, and partial thrombosis were virtually eliminated by heparin delivery from any route. Intimal area 14 days after balloon withdrawal denudation alone was reduced to an equal extent by continuous systemic heparin or by perivascular heparin for the first 3 days. In contrast, endovascular stents produced more exuberant neointimal hyperplasia, the inhibition of which required continuous rather than only early heparin administration. Neither perivascular delivery limited to the first 3 days nor stent-based delivery reduced neointimal hyperplasia as effectively. CONCLUSIONS The antiproliferative and antithrombotic effects of heparin differ markedly, depending on the type of arterial injury and the mode of drug administration. Different forms of injury may require different therapies, and complications of arterial intervention such as excessive neointimal hyperplasia and thrombosis may demand alternate therapeutic regimens. Duration, dose, and site of delivery rather than frank resistance to therapy may explain why experimentally effective antiproliferative and antithrombotic agents fail clinically.

Degranulation of leukocytes in chronic granulomatous disease

Quantitative chemical analyses of the subcellular distribution patterns for acid and alkaline phosphatase, beta glucuronidase and peroxidase were obtained for human peripheral blood leukocytes of four patients with chronic granulomatous disease (CGD). Five young adults with acute infections served as controls. The observations were made on fractions obtained by homogenization and centrifugation of leukocytes previously incubated with or without particles for ingestion. Distributions in resting CGD and normal cells were very similar for acid and alkaline phosphatase and peroxidase, but the proportion of beta glucuronidase in the granule fraction of CGD cells was depressed, with an increased proportion in the soluble fraction. Release of granule-bound enzymes during phagocytosis of a variety of particles was the same for CGD and control cells, except that release of beta glucuronidase was less marked in CGD cells. Total enzymatic activity of CGD cells for the hydrolases studied was normal. The data indicated that granular enzymes are released in a normal fashion in phagocytizing CGD cells. Supportive evidence of release of enzymes into the phagocytic vacuole of CGD cells was obtained by an electron microscopic study of myeloperoxidase.

The adhesive interaction between polymorphonuclear leukocytes and endothelial cells in vitro

The results of this investigation indicate an adhesive specificity between PMN and cultured endothelial cells. This was monitored by the mono-layer collection assay and by direct cell counts using the scanning electron microscope. Both techniques showed that significantly more PMN attached to endothelial cells than to a variety of other cell types. The interaction can be modulated by divalent cations and neuraminidase, implying a role for surface charge. In the presence of chemotactic agents, the number of PMN adhering to endothelial cells increases. This system presents a good model for studying the process of PMN margination which occurs in vivo during the acute inflammatory response.

Vascular leakage induced by horseradish peroxidase in the rat.

Summary Intradermal, intraperitoneal and intravenous injections of horseradish peroxidase solutions induce increased vascular permeability in rats. The vascular leakage is confined to venules, is of short duration, can be inhibited by a combination of histamine and serotonin antagonists and is associated with mast cell degranulation. This phenomenon occurs also in the guinea pig but is minimal or non-existent in the mouse.

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.

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.