Vascular wound healing and neointima formation induced by perivascular electric injury in mice.

Vascular interventions for atherothrombotic disease frequently induce neointima formation, which can contribute to restenosis of blood vessels. As the molecular mechanisms of this process remain largely unknown, quantitative models of arterial injury in transgenic animals may be useful to study this process at the genetic level. Here, an injury model is proposed in which surgically exposed femoral arteries in mice were injured perivascularly via a single delivery of an electric current. Transmission electron microscopy, light microscopy, and immunohistochemistry revealed that electric injury destroyed all medial smooth muscle cells, denuded the injured segment of intact endothelium, and transiently induced platelet-rich mural thrombosis. A vascular wound-healing response resulted that was characterized by degradation of the mural thrombus, transient infiltration of the vessel wall by inflammatory cells, and progressive removal of the necrotic debris. Topographic analysis revealed repopulation of the media and accumulation in the neointima of smooth muscle cells originating from the uninjured borders and progressing into the necrotic center. Within 3 weeks after injury, a neointima of 0.026 +/- 0.003 mm2 (n = 7 arteries) was formed that contained a maximum of 12 +/- 1 layers of smooth muscle alpha-actin-immunoreactive cells. Evans blue staining in five electrically injured arteries revealed a denuded distance of 2.8 +/- 0.2 mm immediately after injury, which became progressively re-endothelialized from the uninjured borders to 2.2 +/- 0.08 mm (P = 0.013 vs freshly injured by analysis of variance), 0.8 +/- 0.22 mm (P < 0.001), and 0.005 +/- 0.003 mm (P < 0.001) within 2, 7, and 14 days after injury, respectively. Analysis of 5'-bromo-2'-deoxyuridine incorporation revealed that a maximum of 35 +/- 10% endothelial cells proliferated within 2 days after injury and that in the media and neointima, a maximum of, respectively, 12 +/- 2% and 18 +/- 3% smooth muscle cells proliferated within 2 weeks after injury. Thus, electric injury of arteries provides a model of vascular wound healing with arterial neointima formation and re-endothelialization that may be useful for the genetic analysis of its molecular mechanisms in transgenic mice.     

Rat pulmonary artery restructuring and pulmonary hypertension induced by continuous Escherichia coli endotoxin infusion

We have studied the effect of continuous endotoxin infusion on rat pulmonary structure and function (69.4 ng/100 gm body weight/min for 24 hours). After 6 days of endotoxin infusion, lack of filling of pre- and intraacinar arteries was evident on pulmonary arteriograms. Microscopy demonstrated lumen narrowing in preacinar arteries and occlusion of intraacinar arteries. Morphometry of patent intraacinar arteries established dilation and increased wall muscle. Widespread alveolar wall injury was evident. After 24 hours of infusion, pulmonary artery pressure was raised (delta 9 mmHg; p less than or equal to 0.001); it then fell but was again increased by day 6 (delta 6 mmHg; p less than or equal to 0.05). Pulmonary vascular resistance was markedly increased at 24 hours (day 0 = 0.1 +/- 0.011 dyne/sec/cm-5; 24 hours endotoxin = 0.572 +/- 0.102 dyne/sec/cm-5; p less than or equal to 0.02). It remained elevated during the infusion period but was not significant. At day 6 the alveolar-arterial oxygen diffusion gradient (A-aDO2) was increased (day 0 = 19.6 +/- 1.39 mmHg, day 6 endotoxin = 33.8 +/- 0.1 mmHg; p less than or equal to 0.001). The arterial oxygen tension (PaO2) was decreased (day 0 = 86.5 +/- 1.8 mmHg, day 6 endotoxin = 74 +/- 2.52 mmHg; p less than or equal to 0.05), as was the arterial carbon dioxide tension (PaCO2) (day 0 = 36.0 +/- 0.73 mmHg, day 6 endotoxin = 30 +/- 1.9 mmHg; p less than or equal to 0.05). Thrombocytopenia occurred during the first 72 hours of infusion (day 0 = 7.41 +/- 0.41 X 10(5)/mm3, day 1 endotoxin = 2.43 +/- 0.30 X 10(5)/mm3, day 3 endotoxin = 2.32 +/- 0.31 X 10(5)/mm3; p less than or equal to 0.001) but by day 6 the platelet count had returned to basal levels (9.9 +/- 0.65 X 10(5)/mm3). Endotoxin increased the number of leukocytes in peripheral blood (day 0 = 12.8 +/- 1.2 X 10(3)/mm3, day 3 endotoxin = 17.0 +/- 1.86 X 10(3)/mm3, day 6 endotoxin = 22.5 +/- 1.8 X 10(3)/mm3; p less than or equal to 0.01 for day 6). Plasma concentrations of 6-keto-prostaglandin F1 alpha decreased during the first 24 hours of infusion (day 0 = 0.56 +/- 0.076 ng/ml, 24 hours endotoxin = 0.27 +/- 0.026 ng/ml; p less than or equal to 0.05) and thromboxane (TX) B2 in the first 15 hours (day 0 = 0.23 +/- 0.058 ng/ml, 15 hours endotoxin = 0.09 +/- 0.14 ng/ml; p less than or equal to 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Prenatal origins of human intrapulmonary arteries: formation and smooth muscle maturation

Recent studies on the morphogenesis of the pulmonary arteries have focused on nonhuman species such as the chick and the mouse. Using immunohistochemical techniques, we have studied 16 lungs from human embryos and fetuses from 28 d of gestation to newborn, using serial sections stained with a panel of antibodies specific for endothelium, smooth muscle, and extracellular matrix proteins. Cell replication was also assessed. Serial reconstruction showed a continuity of circulation between the heart and the capillary plexus from at least 38 d of gestation. The intrapulmonary arteries appeared to be derived from a continuous expansion of the primary capillary plexus that is from within the mesenchyme, by vasculogenesis. The arteries formed by continuous coalescence of endothelial tubes alongside the newly formed airway. Findings were consistent with the pulmonary arterial smooth muscle cells being derived from three sites in a temporally distinct sequence: the earliest from the bronchial smooth muscle, later from the mesenchyme surrounding the arteries, and last from the endothelial cells. Despite their different origins, all smooth muscle cells followed the same sequence of expression of smooth muscle-specific cytoskeletal proteins with increasing age. The order of appearance of these maturing proteins was from the subendothelial cells outward across the vessel wall and from hilum to periphery. The airways would seem to act as a template for pulmonary artery development. This study provides a framework for studying the signaling mechanisms controlling the various aspects of lung development.     

A sequential ultrastructural study of different arteries in the hypertensive rat

Hypertensive vascular disease was induced in rats using weekly injections of deoxycorticosterone and substituting 1% sodium chloride for tap water to drink. The emphasis of the study was on the progression and comparative aspects of alterations produced in the large clastic vessels and the coronary artery.One of the carliest alterations was observed in the intima of all vascular segments. These were characterized by endothelial cell enlargement and a gradual subendothelial accumulation of granular material leading to intimal thickening. In the later stages of the disease, mononuclear cells, fibrin and smooth muscle cells were present within the intima, with the exception of the coronary artery; only smooth muscle cells were seen in the intimal portion of this vessel.Early changes in the media consisted of smooth muscle hypertrophy and widened lamellar units, resulting in increased thickness of the vessel wall. These alterations progressed to include variation in the shape of smooth muscle cells, increasing disorganization of the cellular arrangement, focal cytoplasmic necrosis, and the loss of entire smooth muscle cells.A definite regional difference in susceptibility of individual mural components to hypertensive damage was noted. The most striking feature of this difference was demonstrated by the relatively early, severe involvement of the media of the coronary artery, in contrast to the other arteries examined.These alterations may be initiated by increased intraluminal pressure. In the case of the coronary artery, failure of this vessel to adapt effectively to increased mural tension by structural re-modelling of the arterial wall, may exacerbate these alterations.     

Life history of cutaneous vascular lesions in Sneddon's syndrome.

Sneddon's syndrome is a potentially fatal arterio-occlusive disorder characterized by generalized livedo racemosa and cerebrovascular lesions. Skin biopsies often fail to yield diagnostic arterial lesions. In the present series, affected vessels were found in skin biopsies from 12 of 15 patients with Sneddon's syndrome. Selection of the correct biopsy site (seemingly uninvolved skin at the center of a livedo racemosa area), adequate biopsy size (1 to 2 cm), and serial sections are essential for the detection of relevant vascular pathology. Only small to medium-sized arteries of the dermis-subcutis boundary were found to be involved. Lesions follow a distinct course. The initial stage displays partial detachment of endothelial cells, adhesion of mononuclear cells interspersed with fibrin ("endothelitis"), a marked edema of the surrounding connective tissue with numerous dilated capillaries, and a predominantly lymphohistiocytic infiltrate with polymorphonuclear leukocytes. In the early phase a sponge-like plug is formed from mononuclear cells, fibrin, and red blood cells, leading to partial to complete obstruction. A perivascular inflammatory infiltrate, devoid of polymorphonuclear leukocytes, is located around affected arteries; in the adventitia of the occluded vessel, a very regular corona of dilated capillaries appears that is continuous with more peripheral dilated and branching vessels. Organization of the occluding plug ensues in the intermediate stage by "subendothelial cell proliferation," most likely due to immigrating smooth muscle cells. In the final stage, the occluding artery undergoes fibrosis, shrinkage, and atrophy.     

Smooth muscle cell proliferation in the occluded rat carotid artery: lack of requirement for luminal platelets.

The relationship of intimal smooth muscle cell proliferation in the permanently occluded rat carotid artery to the presence or absence of luminal platelets was examined. Blood was rinsed from the arterial lumen immediately after occlusion and was replaced by autologous, citrated platelet-rich plasma (PRP, 6 to 20 X 10(5) platelets/microliter) or filtered platelet-poor plasma (PPP, less than 100 platelets/microliter). Occluded arteries were studied after 1 to 28 days by light and electron microscopy. Events occurring within the first 2 days included fibrin clot formation, endothelial degeneration and denudation, transmural migration of polymorphonucelar leukocytes and monocytes, and, in PRP-filled arteries, degranulation and disappearance of platelets. By 7 days a neointima was formed by macrophages and undifferentiated cells. The latter cells had some features of vascular smooth muscle cells and were apparently derived from medial cells which traversed the internal elastic lamina. After 14 days, identifiable smooth muscle cells emerged as the predominant cell type in a rapidly growing intimal plaque. No differences could be discerned between arteries originally filled with PRP or PPP. This experimental model is similar to atherosclerosis in dimensions of avascular area and in coexistence of degenerative, inflammatory, and proliferative processes. Cell proliferation deep within an atherosclerotic plaque could be initiated by factors other than platelets, perhaps by products of inflammatory cells.     

Ultrastructure of coronary arteries and myocardium in experimental hypertension

The ultrastructure of coronary arteries and myocardium has been examined after 1–4 weeks of experimental hypertension produced in Wistar-Kyoto rats by unilateral renal artery constriction. Upon sacrifice blocks of ventricular myocardium were processed for light and electron microscopy. Two types of medial necrosis are observed in epicardial arteries: one involving elongated smooth muscle cells throughout the media and the other localized to the outer layers of the vessel wall. Some of the epicardial and intramyocardial arteries exhibit hyperplastic lesions characterized by smooth muscle cells in the subendothelial space. Medial smooth muscle cells of small intramyocardial arteries also exhibit contraction, fragmentation and necrosis. Plasma, cells and fibrin accumulate in the subendothelial space of intramyocardial arteries. Discrete areas of myocardial injury of variable severity are present in the subendocardium of the left ventricle. The vascular lesions in epicardial and intramyocardial arteries are related to the severity of blood pressure elevation and the vasoconstrictor actions of angiotensin II, while the foci of myocardial damage are consistent with ischemic or anoxic injury.     

Acute lung inflammation induced in the rabbit by local instillation of 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine or of native platelet-activating factor.

The intratracheal instillation into rabbits of 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) or native platelet-activating factor (PAF) was shown to induce a dose-dependent acute pulmonary inflammation characterized by accumulation of macrophages in the alveolar space, degenerative and necrotic changes of alveolar epithelium, and accumulation of polymorphonuclear leukocytes (PMNs) and platelets in the alveolar capillary lumens with degenerative changes of endothelial cells. Infiltration of alveolar septa by inflammatory cells and, in a later stage, pulmonary fibrosis were also observed. Intrabronchial instillation of lysoglyceryl ether phosphorylcholine (lyso-GEPC) produced no inflammatory changes or only mild ones. In comparison with acute inflammation induced by intratracheal instillation of C5a des Arg, which is mainly characterized by the presence of neutrophils, red blood cells, and fibrin in the alveolar space, AGEPC and native PAF seem to induce a more severe accumulation of macrophages in the alveolar space and septa and of platelet and PMNs in the lumens of alveolar capillaries. These results are compatible with the concept that during inflammatory reaction an intraalveolar release of PAF contributes to the development of pulmonary injury.     

Hypoxia and incorporation of 3H-thymidine by cells of the rat pulmonary arteries and alveolar wall.

In the pulmonary arterial circulation hypoxia produces increase in thickness of the medial muscle coat as well as of the adventitia; in addition muscle appears in smaller arteries than is normal and the number of small arteries that fill on Micropaque-gelatin injection is reduced. To assess the role of hyperplasia in these changes, the uptake of 3H-thymidine by the cells of the pulmonary arterial wall has been studied in rats exposed to hypobaric hypoxia (exposure to 380 torr) after 1, 3, 5, 7, 10, and 14 days. Using autoradiographs of 1-micron sections, the glutaraldehyde-distended intrapulmonary hilar muscular artery, the peripheral, intraacinar arteries less than 100 micron in external diameter, and the alveolar wall had different patterns of uptake. In the hilar pulmonary artery, after 24 hours of exposure, the labeling index for adventitial fibroblasts is increased eightfold over the control value, and for endothelial cells, threefold, while for medial smooth muscle cells, there is a gradual and small increase to Day 14. Newly muscularized intraacinar arteries are first apparent at Day 3, when they comprise 40% of the intraacinar arteries, increasing to 80% at Day 7. No decrease in density of arteries is found. Uptake of 3H-thymidine by new muscle cells is not apparent until Day 5 when labeling is maximum. The endothelial cells of the newly muscularized arteries show an increased labeling index only at Days 7 and 10. The veins and normally muscular arteries do not show these changes. In the alveolar walls, the concentration of labeled cells is significantly above the control value at Days 3, 5, and 7 and significantly below, at Day 14. At this level, the interstitial, epithelial, and endothelial cells contribute to the increase.     

Studies on the healing of arterial lesions in experimental hypertension

Summary Rats made hypertensive by bilaterally constricting their renal arteries were intermittently given antihypertensive drugs to cause their blood pressure to fluctuate; the intima of their mesenteric arteries was then investigated electron microscopically. The intimal fibrinoid degeneration showed a much weaker tendency to heal than that of the previously reported rats treated continuously with antihypertensive drugs (Kojimahara et al., 1971), and the arteries showed marked dysoria associated with endothelial injury and thrombus formation. Endothelial cells that had migrated from the endothelium into the subendothelial space became intimal cell, which after proliferation by mitosis, formed myofilaments in their cytoplasm, turning into fibroblast-like smooth muscle cells (modified smooth muscle cells) in the intima. Thus some of the smooth muscle cells that proliferated in the intima and took part in the organization of the intimal fibrinoid substance were considered to be derived from endothelial cells.     

Acute cytomegalovirus infection induces a subendothelial inflammation (endothelialitis) in the allograft vascular wall. A possible linkage with enhanced allograft arteriosclerosis.

Clinical and experimental studies have established the accelerating role of cytomegalovirus (CMV) infection on cardiac allograft arteriosclerosis, ie, chronic rejection. We have investigated the mechanisms behind the interaction between CMV infection and chronic rejection. In the first part of the study, 762 endomyocardial biopsy specimens obtained from 47 heart allograft recipients were analyzed. Of these, 28 patients developed CMV infection during the first postoperative year. In 24 of 28 CMV patients, mononuclear inflammatory cells (endothelialitis) were seen in the subendothelium of small intramyocardial arterioles. In CMV-free recipients, only five of 19 had any subendothelial inflammation in the vascular structures P < 0.0001 when compared with CMV patients). The subendothelial inflammation demonstrated an intensive peak at the onset of CMV infection, subsiding slowly thereafter. Morphologically, the inflammatory cells in the subendothelium were small lymphocytes. Only few activated pyroninophilic lymphocytes were seen. Immunohistochemistry revealed that the lymphocytes were mostly T cells (UCHL1+). In the second part of the study, we investigated if a similar endothelialitis could be induced experimentally in allografted rats. We performed rat aortic allografts from the DA (AG-B4, RT1a) donors to the WF (AG-B2, RT1v) recipients and infected the recipients with 10(5) plaque-forming units of rat CMV Maastricht strain 1 day after transplantation. In rat CMV-infected aortic allografts, the frequency of subendothelial leukocyte common antigen (LCA, OX1) positive leukocytes, 1.7 +/- 0.1 (SEM) point score units, was significantly higher when compared to noninfected allografts, 0.8 +/- 0.1 point score units (P < 0.05), and they were most prominent in the intimal space during and following acute infection. During subsequent weeks, the LCA-positive leukocytes were replaced by alpha-actin-positive smooth muscle cells. Instead, most of the cells in intima of CMV-free grafted rats stained positively to alpha-actin from the beginning and were smooth muscle cells. Practically no leukocytes were seen. In rat CMV-infected aortic allografts most subendothelial inflammatory cells represented T cells (W3/25+) and cells of the monocyte/macrophage lineage (OX42+). In conclusion, acute CMV infection is associated with an subendothelial inflammation (endothelialitis) of allograft vascular structures both in human and in rat. Nonactivated T lymphocytes and monocytes predominate the inflammatory lesion in the subendothelium. The results suggest that the virus-linked vascular wall inflammation may play a role in the immune injury toward allograft vascular structures, particularly to endothelium, and thus contribute to allograft arteriosclerosis.     

Expression of endothelin-1 in rats developing hypobaric hypoxia-induced pulmonary hypertension

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Endothelin-1 (ET-1), a 21-amino acid peptide, is a novel and long-lasting vasoconstrictor that increases pulmonary arterial pressure in both in vivo and in vitro experiments. To study the effects of HHE on ET-1 activity in the lungs, 59 male rats were subjected to the equivalent of an altitude of 5500 m for 1 to 4 weeks. In rats exposed to HHE, the mean pulmonary arterial pressure increased significantly from 15.2+/-0.3 (ground level) to 30.6+/-1.5 mm Hg (5500-m level) at 4 weeks, whereas their mean systemic arterial pressure remained normal. The levels of ET-1 mRNA and protein, measured respectively by Northern blot analysis and enzyme immunoassay, increased rapidly in the lungs on exposure to HHE. By in situ hybridization and immunohistochemistry, respectively, ET-1 mRNA and protein were detected in control rats in nonciliated bronchiolar epithelial cells and alveolar epithelial cells, as well as in the endothelial cells of pulmonary arteries, but minimally in the smooth muscle cells of pulmonary arteries. ET-1 mRNA- and protein-reactive smooth muscle cells in pulmonary arteries and ET-1 mRNA-reactive airway epithelial cells were significantly more abundant in rats exposed to HHE than in ground level controls. These results suggest the possibility that in smooth muscle cells in pulmonary arteries and airway epithelial cells, ET-1 may play an autocrine or paracrine role in the remodeling of blood vessels during the development of the pulmonary hypertension that is induced by HHE.     

Ultrastructure of intramural coronary arteries in pigs with hypertrophic cardiomyopathy.

Pigs with hypertrophic cardiomyopathy diagnosed by echocardiographic examination were selected for study from a genetic breeding herd. Under dissecting microscopic examination, intramural coronary arteries in the septum and left ventricular free wall of euthanized pigs were collected for ultrastructural study. The major lesions of wall thickening included degeneration or denudation of endothelium, subendothelial edema, proliferation of collagen fiber, and hyperplasia of smooth muscle cells. Smooth muscle cells proliferated and migrated through the internal elastic lamella into the intima, which caused the early lesion of wall thickening of the intramural coronary arteries. The extent of smooth muscle cell proliferation was related to the severity of endothelial damage. The smooth muscle cells in the intima were identified by immunohistochemical staining (i.e., smooth muscle actin [SMA] stain). Three major types of severe wall thickening with narrow lumen were observed in the intramural coronary arteries. Edema in the intima caused the major lesion of Type I wall thickening. The internal elastic lamella was broken into small interrupted fragments, and fine fragments of elastic fibers surrounded by the cellular processes of smooth muscle were observed in Type I lesions. Many smooth muscle cells proliferated in the intima and media, which constituted the major lesion of Type II wall thickening of the intramural coronary arteries. Many vacuolized, degenerated smooth muscle cells with fewer sarcoplasmic myofilaments could be clearly observed in the Type II lesions. In advanced cases, severe vacuolization and degeneration of smooth muscle cells with the presence of many bizarrely shaped smooth muscle cells in the walls of the intramural coronary arteries could be observed, which caused the major lesion of Type III wall thickening. Pigs with hypertrophic cardiomyopathy, characterized by spontaneously occurring lesions in intramural coronary arteries, may prove a valuable animal model for human disease.     

The origin of subendothelial cells in developing coronary collaterals

Summary Using NPase as a specific and differential marker of endothelial cells and some blood-borne cells, we attempted to provide information on the origin of cells contributing to the formation of the intimal thickening occuring during coronary collateral growth in the dog. NPase reactive endothelial cells, polymorphonuclear and mononuclear blood cells and NPase unreactive smooth muscle cells and modified smooth muscle cells were seen in variable proportions in the subendothelial space during the early period of vascular growth and in the intimal thickening, formed a few weeks later. The medial smooth muscle cell appeared to be the main progenitor of cells present in this zone.The possible transformation of endothelial and blood-formed cells into myointimal cells is discussed. The functional significance of NPase activity in these cells and the further outlook of NPase application as a marker enzyme in the process of atherosclerosis, in experimental hypertension and in various vascular injury processes has been outlined.Supported by Grant 2034 of I.W.O.N.L.     

Ultrastructure of microcirculation in renal grafts in patients with acute or chronic rejections

Using electron microscopy, we studied the renal microcirculation in 31 biopsied specimens from 24 transplants in patients with acute or chronic rejection. Endothelial damage was followed by extensive denudation and platelet attachment, fibrin deposition, focal or whole cytoplasmolysis, rarefaction necrosis of the medial smooth muscle cells, with or without the attachment of inflammatory cells. Medial smooth muscle cells modified by hyperplasia of ergastoplasm, appeared to migrate into the intima and to proliferate there. The endothelium was regenerated with time. Chronic rejection was characterized by "moth-eaten-like" atrophic changes of the medial smooth muscle cells in interlobular arteries with an increase in intercellular spaces with necrotic debris and basement membrane densities. Larger interlobular arteries showed additional mucoid lamellar thickening of the intima, with stenosis. Acute changes in glomerular capillaries included endothelial injury and denudation, thrombotic occlusion and enlargement of subendothelial spaces, apparently due to an increase in permeability. Extensive circumferential mesangial interposition was present in one case of a repeated rejection. Thus, chronicity with various rejections leads to severe luminal narrowing and obstruction in both interlobular arteries and the glomerulus of the grafts.     

ULTRASTRUCTURE OF MICROCIRCULATION IN RENAL GRAFTS IN PATIENTS WITH ACUTE OR CHRONIC REJECTIONS

Using electron microscopy, we studied the renal microcirculation in 31 biopsied specimens from 24 transplants in patients with acute or chronic rejection. Endothelial damage was followed by extensive denudation and platelet attachment, fibrin deposition, focal or whole cytoplasmolysis, rarefaction necrosis of the medial smooth muscle cells, with or without the attachment of inflammatory cells. Medial smooth muscle cells modified by hyperplasia of ergastoplasm, appeared to migrate into the intima and to proliferate there. The endothelium was regenerated with time. Chronic rejection was characterized by "moth-eaten-like" atrophic changes of the medial smooth muscle cells in interlobular arteries with an increase in intercellular spaces with necrotic debris and basement membrane densities. Larger interlobular arteries showed additional mucoid lamellar thickening of the intima, with stenosis. Acute changes in glomerular capillaries included endothelial injury and denudation, thrombotic occlusion and enlargement of subendothelial spaces, apparently due to an increase in permeability. Extensive circumferential mesangial interposition was present in one case of a repeated rejection. Thus, chronicity with various rejections leads to severe luminal narrowing and obstruction in both interlobular arteries and the glomerulus of the grafts.     

Cell-specific nitric oxide synthase-isoenzyme expression and regulation in response to endotoxin in intact rat lungs

Nitric oxide (NO) produced by NO synthase (NOS) serves as a ubiquitous mediator molecule involved in many physiologic lung functions, including regulation of vascular and bronchial tone, immunocompetence, and neuronal signaling. On the other hand, excessive and inappropriate NO synthesis in inflammation and sepsis has been implicated in vascular abnormalities and cell injury. At least three different NOS isoforms (neuronal/brain [bNOS], inducible [iNOS], and endothelial [eNOS]) have been described, which are all expressed in normal lung tissue. We investigated the cell-specific expression of bNOS, iNOS, and eNOS in perfused control rat lungs and lungs undergoing stimulation with endotoxin in the presence and absence of plasma constituents. Lung immunohistochemistry and quantitative evaluation of staining intensity showed endotoxin-induced increase in iNOS expression in particular in bronchial epithelial cells, cells of the bronchus-associated lymphoid tissue (BALT), alveolar macrophages, and vascular smooth muscle cells in a time- and dose-dependent fashion. In endothelial cells, which did not express iNOS at baseline, newly induced iNOS was found in response to endotoxin. In contrast, expression of eNOS was markedly suppressed under endotoxin challenge, particularly in bronchial epithelium, BALT, and alveolar macrophages but also in vascular smooth muscle cells and endothelial cells. eNOS expression in bronchial smooth muscle cells was not altered. In contrast to iNOS and eNOS, cellular expression of bNOS in epithelial cells, nerve fibers, BALT, and endothelial cells did not change in response to endotoxin. All changes in NOS regulation were found to be independent of plasma constituents. We conclude that endotoxin exerts a profound impact on the cell-specific NOS regulation in a large number of lung cell types. Prominent features include de novo synthesis or up-regulation of iNOS, in contrast to down-regulation of eNOS, which may well contribute to vascular abnormalities, inflammatory sequelae, and loss of physiologic functions in septic lung failure.     

ALVEOLAR DESTRUCTION IN EXPERIMENTAL KLEBSIELLA PNEUMONIA

Sequential histological changes of the lungs were studied in experimental Klebsiella pneumonia, using untreated control mice, cyclophosphamide-treated mice, and carrageenan-treated mice. Cyclophosphamide was used to deplete polymorphonuclear leukocytes and monocytes, and carrageenan was used to deplete mononuclear phagocytes selectively. At 72 hours, varying degree of alveolar necrosis could be seen in untreated control mice. However, the lung lesions of cyclophosphamide- or carrageenan-treated mice were significantly different from those of the control mice. The lung lesions of cyclophosphamide-treated mice indicated that destruction of the alveolar septa was not induced by K. pneumoniae itself but by inflammatory cells, because the alveolar walls were preserved very well in spite of considerable bacterial multiplication in alveolar lumina until infiltration of inflammatory cells occurred. The lung lesions of carrageenan-treated mice showed that alveolar spaces were packed with polymorphonuclear leukocytes, but the alveolar walls were preserved very well as far as the authors could tell after examining the lung lesions by silver impregnation staining. These results suggest that macrophages rather than polymorphonuclear leukocytes and organisms play an important role in alveolar injury in experimental Klebsiella pneumonia.     

Alveolar destruction in experimental Klebsiella pneumonia

Sequential histological changes of the lungs were studied in experimental Klebsiella pneumonia, using untreated control mice, cyclophosphamide-treated mice, and carrageenan-treated mice. Cyclophosphamide was used to deplete polymorphonuclear leukocytes and monocytes, and carrageenan was used to deplete mononuclear phagocytes selectively. At 72 hours, varying degree of alveolar necrosis could be seen in untreated control mice. However, the lung lesions of cyclophosphamide- or carrageenan-treated mice were significantly different from those of the control mice. The lung lesions of cyclophosphamide-treated mice indicated that destruction of the alveolar septa was not induced by K. pneumoniae itself but by inflammatory cells, because the alveolar walls were preserved very well in spite of considerable bacterial multiplication in alveolar lumina until infiltration of inflammatory cells occurred. The lung lesions of carrageenan-treated mice showed that alveolar spaces were packed with polymorphonuclear leukocytes, but the alveolar walls were preserved very well as far as the authors could tell after examining the lung lesions by silver impregnation staining. These results suggest that macrophages rather than polymorphonuclear leukocytes and organisms play an important role in alveolar injury in experimental Klebsiella pneumonia.     

Histologic analysis of an immune response in the lung parenchyma of mice. Angiopathy accompanies inflammatory cell influx.

To determine the histologic changes occurring during a pulmonary immune response, the lungs of antigen-primed C57BL/6 mice were examined on various days after intratracheal challenge with 10(8) sheep erythrocytes. The response was characterized by 1) dense perivascular aggregates composed largely of mononuclear cells; 2) endothelial cell hypertrophy and subendothelial inflammatory cell collections in vessels of a variety of sizes; 3) variable degrees of focal, reversible vascular injury (angiopathy) of both muscular arteries and small veins; and 4) increased cellularity of alveolar walls. Inflammatory cells appeared to emanate from small veins and venules and from minute thin-walled vessels adjacent to large arteries. The reaction peaked at 3 to 4 days and then gradually declined over a period of 6 weeks, never quite reaching baseline. We believe that this experimental model will be an important means of further defining both the mechanisms of lymphocyte entry to the lungs in response to antigen and the factors controlling the pathogenesis of related angiopathies.