Exp Toxic Pathol 2000; 52: 308-311URBAN & FISCHERhttp://www.urbanfischer.de/joumals/exptoxpath

Novartis Pharma AG, Preclinical Safety, Pathology, Basle, Switzerland

Pulmonary vascular sclerosis in an albino rat with leukemia

V. PACE, A. T. MAHROUS, and E. PERENTES

With 5 figures

Received: June 20,1999; Accepted: July 19, 1999

Address for correspondence: Dr. med. vet. V. PACE, Novartis Pharma AG, Preclinical safety, Pathology, WSH­2881.4.05, CH - 4002 Basle, Switzerland; Fax: +41 61 3 24 15 70, e-mail: virgilo.pace@pharma.novartis.com

Key words: Pulmonary vessels; Lung vessels; Vascular sclerosis, lung; Leukemia; pulmonary vascular sclerosis; Emboli,pulmonary; Lung emboli; Pulmonary emboli.

Summary

The animal investigated was a two years old male con­trol Sprague-Dawley rat which died spontaneously duringa carcinogenicity study. Post-mortem examination dis­closed hepatic and splenic enlargement. At microscopicalexamination, massive leucaemic infiltration was observedin many tissues/organs, including bone marrow, spleen,liver and renal blood vessels. A very unusual finding wasobserved in the lung, consisting of scattered micronoduleswhich replaced most of the lung parenchyma. They con­tained collagen, displaying a somewhat circular distribu­tion at the periphery of the lesions, fibrin, leukemic cellsand fibroblasts.

Immunostaining for desmin revealed the presence ofsmooth muscle fibers within the nodules, while stainingfor elastic fibers showed clearly that the internal and exter­nal elastic membranes were identifiable within the nod­ules. The diagnosis of pulmonary vascular sclerosis wasmade on the basis of microscopical and immunohisto­chemical findings.

Introduction

The pulmonary circulation is characterized by low re­sistance and low blood pressure. Pulmonary hyperten­sion is mainly due to an increase in pulmonary vascularresistance and is, most frequently, related to chronic ob­structive or interstitial lung diseases, recurrent pul­monary emboli, or antecedent heart disease. Less fre­quently, pulmonary hypertension is encountered in pa­tients in whom all known causes of increased pulmonarypressure are excluded, and this is referred to as primary

308 0940-2993/00/52/04-308 $ 12.00/0

or idiopathic pulmonary hypertension (4). Dysfunctionof pulmonary vascular endothelial cells seems to play acentral role in the vascular responses of both idiopathicand secondary pulmonary hypertension (12). In animals,the leguminous plant Crotalaria spectabilis, which con­tains the alkaloid monocrotaline, causes hypertensionand pulmonary vascular lesions (1, 10, 11).

Materials and methods

In the present investigation, the affected animal was atwo years old male control Sprague-DaWley derived rat(strain Tif:RAI) from a longterm study conducted underbarrier maintenance conditions. The rat died spontaneous­lyon day 720 of the experiment and a complete necropsywas performed. At necropsy, enlargment of the liver andspleen was noted. All tissues were preserved by immersionin neutral buffered 4% formaldehyde solution. Specimensof a standard list of organs and tissues were embedded inparaffin, cut at 5 /lm and stained with hematoxylin andeosin (HE). Additional stainings were performed for thelung, including Van Gieson for collagen, silver impregna­tion for elastic fibers, periodic acid-Schiff reagent (PAS)and the immunohistochemical reaction for desmin. For thelatter, 5 /lm thick paraffin sections were immunostainedusing a mouse monoclonal anti-desmin antibody (DakoCo., Carpinteria/CA, USA, lot 045, dilution 1:800). Theimmunohistochemical reaction was carried out by usingthe streptavidin/peroxidase method (Dako DiagnosticsAG, Zug, Switzerland, dilution 1: 100). Prior to the im­munohistochemical procedure, sections were placed in a10 mM citrate buffer solution (pH 6.0) and heated 2 x 5minutes in a microwave processor at 95°C.

Results

At microscopical examination, massive leukemic in­filtration (probably of myeloid origin) was observed inseveral organs/tissues, including bone marrow, spleen,liver and the renal blood vessels. Most of the neoplasticcells had a blastic appearance but some had anular orlobed form. Peculiar lesions were seen in the lung. Thisorgan exhibited an widespread dissemination of micro­nodular formations composed of concentrically arrangedcollagen fibers, fibrin, leukemic cells and fibroblasts(figs. I and 2). These lesions were devoid of inflamma­tory cells. A lumen, reflecting probably recanalization,was present in several nodules (fig. 2). The immuno­staining for desmin revealed the presence of smooth

muscle fibers within nodules (fig. 3); their morphologyclearly suggested a vascular origin. This was furtherconfirmed by the silver impregnation for elastic fiberswhich disclosed, within the micronodules, the presenceof thickened and delaminated internal and external elas­tic membranes corresponding mainly to medium sizedarteries (fig. 4). The PAS reaction showed PAS-positivedeposits indicating the presence of fibrin in the lumenand in the wall of the affected vessels, and the VanGieson staining (fig. 5) confirmed the presence of vari­able amount of collagen fibers in the vascular wall. Ad­ditional lung lesions included small areas of alveolaremphysema, mild interstitial fibrosis, focal hemorrha­gies and alveolar macrophages loaded with hemosiderin.On the basis of the above findings, the diagnosis of pul-

Fig. 1. Lung; rat. Pulmonary vascular sclerosis. Large number ofmicronodular formations disseminated in the lung (arrows). HE x20.Fig. 2. Lung; rat. Same area as in figure I; higher magnification.Lumen and recanalization (arrows) associated with the presence ofsclerosis of the vascular wall (arrowheads). HE xIOO.Fig. 3. Lung; rat. Positive immunoreaction for desmin indicating thepresence of smooth muscle within the micronodules (arrows), andsuggesting the vascular involvement in the lesion. Streptavidinlper­oxidase method for desmin, hematoxylin counterstain, x 100.Fig. 4. Lung; rat. Silver impregnation for elastic fibers revealingthickening and delamination of the internal and external elastic mem­branes of the arteries (arrow). Silver impregnation, x160.Fig. 5. Lung; rat. Van Gieson staining showing the presence of colla­gen fibers in the wall of the vessels (arrowheads). Van Gieson, x90.

Exp Toxic Pathol 52 (2000) 4 309

monary vascular sclerosis was made. Although otherworkers (7, 8) claimed immuno-positive results in ratendothelial cells with anti-human Factor VIII-relatedantigen antibodies, in our hands (results not shown) nonconsistent results were obtained in rat endothelial cells,while intense immunoreactivity was found in humanand canine formalin-fixed tissues. These results are fur­ther supported by the manufacturer's specifications (e.g.Dako, Glostrup, Denmark) where it is clearly indicatedthat no cross-reaction with rat endothelia can be demon­strated on formalin-fixed tissues. Therefore, for theabove reasons, in the present investigation it was decid­ed not to use anti-human Factor VIII related antigen an­tibodies for diagnostic purposes. In addition to the lunglesions, myocardial hypertrophy of the right ventricle,associated with a mild vacuolar degeneration and in­flammatory cell infiltration of both ventricles, was alsopresent.

Discussion

Secondary pulmonary vascular hypertension is rela­tively frequent in man. It is frequently secondary tochronic obstructive or interstitial lung diseases, recur­rent pulmonary emboli, and antecedent heart disease(12). The consequence of pulmonary vascular hyperten­sion is the cor pulmonale (pulmonary heart disease).Two forms of cor pulmonale are seen: acute and chronic.Acute cor pulmonale refers to the right ventricular di­latation that follows massive pulmonary embolization.Chronic cor pulmonale usually implies right ventricularhypertrophy. Various vascular lesions lead to chronic corpulmonale: repeated small pulmonary embolizations orany form of diffuse pulmonary vascular involvement,such as encountered in systemic hypersensitivity states,may cause pulmonary hypertension. Any chronic lungdisease may lead to cor pulmonale, either by increasingpulmonary vascular resistance or by the induction of in­trapulmonary vascular shunts; this includes chronic ob­structive pulmonary diseases, pneumoconioses, sar­coidosis, idiopathic interstitial fibrosis, bronchiectasisand pulmonary or mediastinal tumors. In all these pul­monary parenchymal disorders, polycythemia and thevasoconstrictive effects of hypoxemia and respiratoryacidosis contribute significantly to the development ofthe pulmonary hypertension. Dysfunction of pulmonaryvascular endothelial cells plays a central role in the vas­cular responses of both idiopathic and secondary pul­monary hypertension. In secondary forms of pulmonaryhypertension, endothelial cell dysfunction is producedby the process initiating the disorder, such as the me­chanical injury associated with left to right shunts or thebiochemical injury produced by fibrin in thromboem­bolism (12). In idiopathic pulmonary hypertension,endothelial dysfunction and injury occur, idiopathic inmost cases, but sometimes associated with autoimmunedisorders, toxic substances, and perhaps specific genetic

310 Exp Toxic Pathol 52 (2000) 4

determinants. Decreased elaboration of prostacyclin, de­creased production of nitric oxide, and increased releaseof endothelin all promote pulmonary vasoconstriction(12). Pulmonary hypertension can also be associatedwith drugs and toxins. Following the administration ofcompounds like Aminorex® (known as sympathetic cat­echolomines) by a parenteral route, smooth muscle cellnecrosis occurs in the media of the vessels (arteriolesfrom 40 to 600 ~m in diameter are predominantly affect­ed); thereafter, a full range of lesions from medial hyper­trophy and intimal fibrosis to perivascular inflammationand plexiform lesions occur. Secondary phenomena ofhyalinization of arterial walls, fibrin deposition, organi­zation and recanalization are superimposed (2, 4). Vaso­constriction has been inferred as morphologic, as well asphysiologic, grounds to contribute to the pulmonary hy­pertension. Attempts to reproduce the disorder in experi­mental animals by administering Aminorex® or cate­cholamines have been consistently unsuccessful (3).Similar pulmonary vascular injuries can be induced bythe alkaloid monocrotaline. When ingested, this planttoxin, is bioactivated in the liver by cytochrome P450monooxygenases to a reactive pyrrolic derivate, proba­bly monocrotaline pyrrole (9). The lung injury is charac­terized by structural remodelling of pulmonary bloodvessels (including degenerative, inflammatory and pro­liferative changes) and by an increase in pulmonary arte­rial pressure (11). Since similar structural remodellingalso occurs in lungs of people with certain forms ofchronic pulmonary hypertension, the monocrotaline­treated rat has been used as an animal model for humanpulmonary vascular disease (II, 13). Furthermore, me­dial smooth muscle necrosis is induced after administra­tion of certain pharmaceutical agents such as vasodila­tors and inotropics. These agents are known to inducearteriopathies in dogs and rats, although at differentsites. In dogs, the coronary arteries are mainly affected(5), whereas in rats the mesenteric arteries are the princi­pal sites of injury (6, 14). Since these agents are diversein structure and biochemical activity, the mechanism oftoxicity appears to be related to their pharmacodynamicactivity rather than to a direct toxic effect.

In the present case, pulmonary vascular sclerosis, inthe form of nodular lesions, was consequent to increasedblood viscosity and to repeated embolizations ofleukemic cells in small pulmonary vessels, which, intum, increased vascular resistence and induced pul­monary hypertension and, as consequence of thesehemodynamic changes, degeneration and sclerosis ofthe arterial wall occurred. Like in man, chronic cor pul­monale in the form of right ventricular hypertrophy wasalso present. To our knowledge, this is the first time thatsuch lesions are reported in rat.

Acknowledgements: We thank Mrs. N. VIDOTIO, Mrs.A. VIGNUTELLI and Mr. F. DA SILVA for histologic prepara­tion and staining of tissues.

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