G. Stoica,* J. Hoffman,* and P. H. YuentFrom the Department of Veterinary Pathology, College ofVeterinary Medicine, TexasA &M University, CollegeStation, Texas*; and the University ofTexas SystemCancer Center, Science Park-Research Division,Smithville, Texast
Moloney murine sarcoma virus (MoMuSV349) isproduced byMuSV349 cells in at least eight-fold ex-cess over the replication-competent helper virus.'Less than 48-hours-old Balb/c mice inoculated in-traperitoneally with supernatant from MuSV349cells containing i-14 MuSV349 infectious unitsdeveloped clinical symptoms, including severegen-eralized wasting, 15 to 20 days after inoculation.These infected mice became moribund 35 to 45days after inoculation. Gross examination of thebodies revealed thepresence ofcutaneous and sub-cutaneous 0.2-cm to 1.5-cm macules, plaques, ornodules located predominantly on the ventral ab-domen and legs. Nodules also were found in thespleen, liver, ovaries, testes, meninges, nerves, andskin. The nodules were semisoft, cystic, orsolidandsome expressed variable amounts of blood. Histo-logic examination of the macules, plaques, andnodules showed spindlelike cells intermingled withtortous, jagged vascular channels lined by plumpand normal endothelial cells and unlined slitlikespacesfilled with erythrocytes. These angiomatouslesions were infiltrated extensively with neutro-phils, lymphocytes, macrophages, and someplasma cells. In some cases the lesions also in-cludedfoci ofdenselypacked eosinophils. These an-giomatous lesions are clearly distinguishablefromthe fibrosarcomas induced by the myeloprolifera-tive sarcoma virus (MPSV), and resemble the sar-comas induced in mice by Gz-MSVand Balb MSV,the sarcomas induced in rats byMPSVandHa-MSV,and the acute generalized form of Kaposi's sar-coma (KS) associated with acquired immune defi-ciency syndrome (AIDS) in humans. Electron mi-croscopy also revealed the presence of numerousextracellular type C virions and virions buddingfrom the plasma membrane of endothelial and
spindlelike cells. Erythrophagocytosis by the endo-thelial and spindlelike cells was demonstrated bylight and electron microscopy. The widely dissemi-nated lesions appear to have developed simulta-neously as a consequence of viremia rather thanmetastasis. (AmjPathol 1990, 136:933-947)
The Moloney murine sarcoma viruses (MoMuSVs) consti-tute a family of viruses distinguished from all other retrovi-ruses by the incorporation of the mouse mos gene intotheir genomes. The first MoMuSV was isolated from theplasma of a Moloney murine leukemia virus (MoMuLV)-infected Balb/c mouse with a rhabdomyosarcoma.2 Sinceits isolation, a number of biologically or genetically distin-guishable MoMuSV strains have been identified. Theseinclude HT-1 ,3 ml, m3,4'5 myeloproliferative sarcoma virus(MPSV),6'7 Gazdar (Gz)-MSV,8 MuSV124, MuSV349,15F,9 MuSV124 clone 14,10 SV7,1 and tsllO. TheMuSV124 clone 14 is a spontaneously generated deletionmutant of MuSV12410 and ts1 0, and is a temperature-sensitive derivative of MuSV349.11 Among these Mo-MuSV isolates, only the Gz-MSV was isolated from aspontaneous tumor. It appears therefore that the first Mo-MuSV isolate and Gz-MSV may have arisen separatelyas the result of two independent recombination eventsbetween the genome of MoMuLV and a normal mousemos gene.12 Genetic differences among the remainingMoMuSV strains appear to have arisen as a result of pointmutations, deletions, and insertions subsequent to thetransduction of c-mos.
The genome size of the various MoMuSVs vary fromabout 3.6 kb for MuSV124 clone 1410 to about 7.0 kb forMPSV.13 By heteroduplex mapping13'12'14 of the MuSV ge-nome with that of MoMuLV, restriction endonucleaseanalysis, and DNA sequencing, it was shown that in eachof the MuSV examined, the -1.2 kb cellular mos se-quence replaces most of the MoMuLV env and pol genes,while retaining varying amounts of the gag gene.
Supported by the University of Texas M.D. Anderson Cancer Center,Houston, Texas New Program Development Fund, and a grant from theInstitute of Biotechnology, Texas A & M University.
Accepted for publication December 6, 1989.Address reprint requests to P. H. Yuen, University of Texas System
Cancer Center, Science Park-Research Division, Smithville, TX 78957.
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934 Stoica, Hoffman, and YuenAJPApril 1990, Vol. 136, No. 4
The size of the mos and gag products of MuSV1 24,ml, HT-1, and MPSV differ15-20 (and Wong, Soong, andYuen: Unpublished data). However, these differences donot appear to be important for transformation of fibro-blasts in vitro. Their effect on oncogenicity in vivo has notas yet been determined. However, the long terminal re-
peats of MPSV have been shown21 to be an importantgenomic determinant responsible for the induction of my-eloproliferative disease in adult DBA/2 mice.
All MoMuSV strains tested in mice or rats induced sar-
comas (ie, tumors of mesenchymal origin). The tumorsinduced by early passages of MoMuSV have been vari-ously described as rhabdomyosarcomas,2 spindle cellsarcomas, undifferentiated sarcomas, and atypical granu-
lomas, with a small proportion developing into mesenchy-mal cell sarcomas and hemangiosarcomas.62324 Thiscould be due to the heterogeneity of the virus inoculaused in these studies, since several genetically or biologi-cally well-defined variants have subsequently been iso-lated from the original MoMuSV-MuLV complex. Gazdar-MSV, which has probably arisen in a separate recombina-tion event,12 was reported to induce in rodents spindlecell sarcomas with a prominent angiomatous compo-
nent.8Apart from MPSV, the pathology induced by the vari-
ous MoMuSV derivatives of the original MoMuSV isolatehave not been investigated. Myeloproliferative sarcoma
virus is unique among these MoMuSVs in that, in additionto its ability to induce fibrosarcoma, it also induces prolif-eration of both erythroid and myeloid cells in both adultBALB/c and DBA/2 mice.7,25,26 Consequently, studies7 onMPSV have focused on the characterization of this aspectof the disease. Recently MPSV was also shown27 to in-duce not only myeloproliferative disease but also heman-giosarcomas in the spleen, cerebellum, and spinal cordof Jar-2 rats. Although it has been shown that neither mlrescued by MoMuLV or Friend (F)-MuLV nor MuSV1 24released by G8-124 cells9 induced myeloproliferation,','whether sarcomas were also induced by MoMuSV 124or ml was not reported. To determine whether differentstrains of MoMuSV also differ in the solid tumors they in-duce, we compared the disease manifested by MuSV349released from TBMuSV349 cells19 with that of MPSV res-
cued by MoMuLV from MPSV DNA-transformed NIH/3T3cells following intraperitoneal injection into <48-hour-oldBalb/c mice. We observed that, in contrast to MPSV,MuSV349 did not induce myeloproliferative disease. Theprimary lesions induced by MuSV349 were angiomatouslesions. These lesions were disseminated throughout thebody. They were primarily characterized by 1) proliferat-ing vascular channels lined by normal and plump endo-thelial cells and unlined slitlike spaces filled with erythro-cytes, 2) uniform, undifferentiated spindlelike cells sur-
rounding the vascular components, and 3) diffuse
inflammatory infiltrate consisting of neutrophils, macro-phages, and eosinophils. In histopathology, these lesionsresemble the hemangiosarcomas induced in mice by Gz-MSV and Balb MSV,-" the hemangiosarcomas induced inrats by MPSV,27 and Harvey murine sarcoma virus (Ha-MSV),3031 and the acute, generalized form of Kaposi'ssarcomas observed in AIDS patients. We also confirmedearlier findings7'25 26 that MPSV induced erythroid and my-eloid proliferation in the spleen as well as fibrosarcomas.The histopathology of these fibrosarcomas is in agree-ment with published reports.7
Materials and Methods
Viruses
TBMuSV349 cells produce MuSV349 in at least an eight-fold excess over the helper virus.1 About 1 4 MuSV349focus-forming units (FFU) in 0.1 ml medium were inocu-lated intraperitoneally into <48-hour-old Balb/c mice.Cloned MPSV DNA (p18-663) was obtained from Dr. W.Ostertag. The DNA was transfected into NIH/3T3 cells. Atransformed focus was isolated, expanded, and MPSVwas rescued by superinfection with MoMuLV. About 1 4MPSV FFU together with MoMuLV, in 0.1 ml medium wereinjected intraperitoneally into Balb/c newborn mice.
Virus Assays
The 1 5F and focus-forming assays have been describedpreviously.32 For the focus-forming assay, NIH/3T3 cellswere used instead of TB.
Animals
The Balb/c mice were bred from a breeding pair pur-chased from the National Institute of Health (NIH).
Histologic Study
Twenty-eight mice were injected with supernatant fromTBMuSV349 cells,28 five mice were injected with MPSVrescued by MoMuLV, and three uninfected mice servedas controls. All mice exhibited severe clinical signs 35 to45 days after inoculation. Those used for hematologic andelectron microscopic studies were killed by intraperitonealoverdose of pentobarbitol. The remainder were killed bycervical dislocation. The controls were killed at the end ofthe experiment.
A complete necropsy was performed in every caseand tissues saved in phosphate-buffered 10% formalin.Tissues were slowly decalcified as necessary, dehy-drated, embedded in paraffin, sectioned at 5 A, andstained with hematoxylin and eosin (H&E). Other specialstains were used when necessary.
Hematologic Study
This study was performed prior to euthanasia on 10MuSV349 infected and 3 control mice. Mice were anes-thetized with pentobarbitol and blood collected with hepa-rin by heart puncture. Blood smears were performed im-mediately, as were the total red and white blood cellcounts (Coulter Electronics, Hileah, FL). Smears werefixed with methanol and stained with Leishman's. The redblood cells and leukocytes were evaluated and quanti-tated microscopically.
Electron Microscopy
Tissues for microscopic evaluation were selected fromfive MuSV349 infected mice that developed Kaposi's sar-coma (KS)-like nodules on the peritoneal surface. Micewere killed by an intraperitoneal overdose of pentobarbi-tol, then perfused through the aorta, via the left ventricle,with 50,ul of heparin in phosphate-buffered saline (PBS),(pH 7.4, 330 mOsm, 370C), followed by Karnovsky fixa-tive. Sections of the KS-like lesions were cut into 1.0-mmcubes, washed twice in cacodylate buffer, and postfixedin 1.3% osmium tetroxide in s-collidine at pH 7.4. Tissueswere dehydrated through ascending concentrations ofethanol, transferred to propylene oxide, and embeddedin epon. Thick sections were cut at 1 ,u with glass knivesand stained with toluidine blue (1%). Tissue blocks se-lected for ultrastructural evaluation were cut on an LKBultramicrotome (Pharmacia LKB Biotechnology Inc., Pis-cataway, NJ) with diamond knives. Thin sections werestained with uranyl acetate and lead citrate, and exam-ined with a Zeiss electron microscope.
Lectin Staining
Tissues with KS-like lesions from MuSV349- and MPSV-infected tissues with fibromatous nodules and tissuesfrom uninfected mice were fixed in 10% buffered formalin,embedded in paraffin, and sectioned at 5 g. These weredeparaffinized and rehydrated through graded alcohols,then washed in PBS and incubated with fluorescein iso-thiocyanate (FITC)-labeled Ulex europeus lectin 1 (UEA-1, Vector), diluted in PBS to a concentration of 50 ,ug/ml
for 30 minutes. The stained sections were washed withPBS five times and coverslipped with glycerin solutioncontaining 1% phenylenediamine. The control sectionswere incubated in a solution that contained, in additionto the FITC-labeled lectin, an excess of the appropriateinhibitory carbohydrate (alpha-L-fucose). Results wereconsidered positive only in the absence of staining of con-trol sections. The stained tissues were examined with anOlympus BHA fluorescence microscope (Olympus, To-kyo, Japan) equipped with a 100-Watt mercury bulb forepifluorescence illumination with an FITC interference ex-citation filter, a DM 500 dichroic mirror, and a 0-515 barrierfilter.
Results
Clinical Symptoms Induced byMuSV349 and MPSV
All the MSV349-infected mice weighed 30 to 50% lessthan their control counterparts when they were killed 35to 45 days after inoculation. Clinical signs were apparent15 to 20 days after inoculation. Kyphosis, cyanotic mu-cous membranes, exophthalmia, piloerection, and loss ofappetite were observed during the last 12 to 16 hours oflife. The experimentally infected mice exhibited a concur-rent wastinglike syndrome. Myeloproliferative sarcomavirus-infected mice showed similar clinical symptoms.
Hematologic findings revealed neutrophilia and lym-phopenia in MuSV349-infected mice (Table 1).
Gross Pathology Induced byMuSV349 and MPSV
The organ distribution of angiogenic lesions in MuSV349-infected mice is depicted in Figure 1. The MSV349-in-fected mice exhibited subcutaneous, blue-red macules,plaques, and nodules, ranging in size from 0.2 to 1.5 cmin diameter, and found predominantly on the ventral abdo-men and legs (Figures 2, 3). Multiple blue to white firmnodules (0.2 to 1.5 cm) also were observed in the abdomi-nal muscles, retroperitoneum, mesentery, spleen, ribs,stomach, intestine, lungs, ovaries, and testes. In cross
Table 1. Circulating White Cell Counts at theTermination Period in Control andMSV349-inoculated Mice
Virus
ControlMSV349
No. ofanimals
35
Segmented %(mean ± SEM)
33±2.776 + 1 .7
Lymphocytes %(mean± SEM)
66±2.624 ± 0.5
936 Stoica, Hoffman, and YuenAJPApril 1990, Vol. 136, No. 4
100 -
80 -
0 0
0 =5 c
c E :
0
E
H0_
$CDM> s° E zzCL >v E 0 0 0
0~~~0. :2 E
o~~~~~~~~~~~~~~~~ co0
ORGAN/TISSUE co 0E CD0 0
Figure 1. Organ/tissue distribution ofKS-like lesions oJ'MoMuSV349-infected newborn Balb/c mice.
section, these nodules were semisoft or cystic to solid,with some containing variable amounts of blood.
In some mice, the muscles of the thigh showed prolif-erative white-blue masses (1 cm in diameter) that ex-
tended to the hock joints. The lungs exhibited multiple redpatches, which were disseminated throughout the paren-
chyma. A somewhat interesting finding was the gross ap-
pearance of the ovaries, which exhibited bilateral, uni-form, moderate enlargement and red discoloration. Exam-ination of the testes demonstrated multiple miliaryhemorrhagic nodules disseminated predominantly on thetunica albuginea. The MPSV-infected mice also showeda similar distribution of solid tumors in multiple organs, butthese were white, firm nodules.
Histopathology Induced by MuSV349
Microscopic examination of the MuSV349-induced angio-matous lesions grossly described above showed thateach lesion is composed of two main elements: 1) prolifer-ating irregularly shaped vascular channels lined by normalor plump endothelial cells and slitlike spaces filled witherythrocytes. The plump endothelial cells sometimes formpapillae that extend into and occasionally obliterate thelumen of the vascular channels; and 2) spindlelike, some-
what pleomorphic cells with prominent oval nuclei pack-ing the intervascular spaces. Occasionally, the plump en-
dothelial and spindlelike cells exhibited atypical mitosis,intracytoplasmic and interstitial hyaline bodies, and accu-
mulation of hemosiderin pigment. The preponderance ofthe two main elements varied with the lesion. For subcuta-neous and visceral tumors, proliferating endothelial cellspredominated (vascular pattern), but in the large muscletumors, the spindlelike cells predominated. All these KS-like lesions were extensively infiltrated by neutrophils, lym-phocytes, plasma cells, and macrophages. They alsocontained a few syncytia. In some lesions, foci of denselypacked eosinophils were also observed. In addition,some of the KS-like lesions exhibited benign and othersmalignant characteristics. No vascular metastatic throm-boemboli was detected. The lesions were most likely mul-tifocal in origin. However, the possibility that some were
metastases cannot be excluded.
Skin
Multiple, diffused, or localized areas of intracutaneoushemorrhage, not usually associated with tumor nodules,were frequently seen, especially in the leg and back. Anapparent increase in vascularization around the hair folli-cles and adnexal structures were often present in the ear-
liest lesions (promontory signs) (Figure 4). The mostprominent lesions were the KS-like plaque and nodules.In cross section (Figure 5), these were characterized by
Figure 4. A section of haired skin exhibiting a fully developed patch ofKS-like lesion, characterized by irregularly shaped vessels(arrows), proliferation ofspindlelike cells and infiltration by inflammatory cells. Note (P) the widely dilated, irregularly shapedvascular channel, and a section ofthefollicle protruding into the vascular space ('promontory"sign). H&E X260. Figure 5. Highermagnification ofa section of a KS-like lesion from the skin ofan infected mouse. Note the irregularly shaped vascular channels(arrow) and slitlike spaces (small arrow) containing erythrocytes, and lined byfattened endothelial cells. The intervascular spaceis occupied by spindlelike cells admixed with inflammatory cells. A slitlike space can also be seen around a pre-existing vascularchannel. H&EX420.
irregularly shaped vascular channels of various sizes, slit-like spaces containing erythrocytes and interspersed withspindlelike cells, neutrophils, macrophages, and eosino-phils. The channels are lined by normal and atypical endo-thelial cells, which occasionally formed multiple layers andintraluminal ingrowth. The histologic appearance of thesechanges in the skin was suggestive of macules, plaques,and nodular-type lesions of Kaposi's sarcoma.33
Musculoskeletal System
Kaposi's sarcoma-like lesions usually developed aroundthe xiphoid and expanded over the ventral region of therib cage as coalescing hemorrhagic nodules. The para-
...
vertebral region also showed numerous similar lesionsthat appear to have arisen from the periosteum and infil-trated the adjacent muscles. Kaposi's sarcomalike nod-ules also were found in the musculature of the thigh andabdomen separating and replacing the muscle fibers (Fig-ure 6). In addition, neoplastic cells often showed intracy-toplasmic hyaline bodies and erythrophagocytosis (Fig-ure 7). This latter feature is a characteristic of KS-like le-sions in many of the affected organs. There was no boneinvolvement in any of the cases.
Ovaries and Testes
In the ovaries, KS-like lesions were found distributedthroughout the parenchyma, intermingling with normal fol-
fi *w ~..a..- }8x'K...........+_-x.............;s4 ...._Figure 6. Appearance ofan angiomatous KS-like lesion within the thigh muscle. Notice the irregularly shaped vascular channels(arrow) lined by normal endothelial cells and small neovessels and slitlike spaces (open arrowheads). The musclefibers (M) areseparated, fragmented and replaced byproliferating spindlelike cells and neutrophils. (H&E X 140). Figure 7. Higher magnificationofan area depicted in Figure 6 (solid triangle) showing many hyaline globules (small arrows) and erythrocytes within the cytoplasmof a neoplastic cell (large arrow). Note area of spindle cell proliferation (S) and slitlike spaces containing erythrocytes (thickarrows). H&EX610.
II
940 Stoica, Hoffman, and YuenAJPApril 1990, Vol. 136, No. 4
I
Figure 8. Colon (C): Submucosal KS-like nodule consisting of irregularly shaped vascular channels of various sizes (arrows).H&EX 120. Figure 9. Appearance ofa KS-like nodule within thepancreas. Afocus ofspindle-like cells (S) replaces the normal exocrinepancreatic acini. Notice the presence ofirregularly shaped vascular channels (arrows), slitlike spaces (L) lined byflattened endo-thelial cells and also some inflammatory cells. H&E X260.
licular structures in various stages of development. In ad-dition, the follicular granulosa cells showed moderate tosevere degeneration and necrosis characterized bynuclear pyknosis and vacuolated cytoplasm.
In the testes, the KS-like lesions observed were primar-ily miliary hemorrhagic nodules localized on the tunica al-buginea. Similar lesions occasionally were found in thetesticular pampiniform plexis, but none were observed inthe parenchyma.
Gastrointestinal Tract
type of lesions were detected as white to hemorrhagicnodules on the serosal surface. The mucosa was not in-volved.
Pancreas
Kaposi's sarcomalike nodules of variable size were foundin the exocrine pancreas (Figure 9). Acinar necrosis andsynctia were observed in the exocrine pancreas, but nolesions were detected within the islets of Langerhans.
Kaposi's sarcomalike lesions were found primarily in thelamina propria and submucosa of the stomach and colon.In two mice, the growths had involved the mucosa, laminapropria, submucosa, tunica muscularis, and serosa of thestomach and colon (Figure 8). The proliferation in thesetwo cases may have started within the lamina propria andfrom there spread to form sizable nodules (1.5 cm in diam-eter) on the serosal surface. In the small intestine, these
Liver
Usually, KS-like lesions were observed on the capsule ofthe liver as multifocal nodules. In two cases, lesions wereobserved within the parenchyma. In one of the cases, aKS-like lesion was found surrounding the periportal areaand consisted of irregularly shaped vascular channelslined by normal endothelial cells in a bed of spindlelike
Figure 10. Section ofa spleen showing an angiomatous lesion (A) and normal appearance oftheparenchyma. Inset shows the highlyvascularized nature of the lesion, plump endothelial cells (arrow) lining a poorly defined vascular lumen and spindlelike cells.H&E X 120, inset X260.
cells and neutrophils. In the second case, the lesion con-
sisted of spindlelike cells only.
Spleen and Lymph Nodes
Splenic architecture was preserved (Figure 10). Most ofthe KS-like lesions were localized pericapsularly andwithin the parenchyma. Occasionally there was a mild fol-licular hyperplasia and a slight increase in extramedullaryhematopoiesis in the red pulp. In the lymph nodes of theabdominal and thoracic cavities, KS-like lesions also wereobserved under the capsular layer and within the paren-
chyma. All the lymph nodes examined exhibited variabledegrees of reticuloendothelial hyperplasia. The bone mar-
row did not have any angiomatous changes or myelofi-brosis.
Kidney
In the kidney, similar lesions as described above were
found pericapsularly. In one mouse, the renal tubular epi-thelial cells of the renal medulla were transformed. Thistransformation was characterized by loss of cell polarityand normal tubular arrangement, intense cytoplasmic ba-sophilia, and numerous mitoses.
The lungs of infected mice manifested an interstitial pneu-monitis consisting of mild to moderate infiltration of thealveolar septae with band and ring neutrophils, lympho-cytes, and macrophages. There also were multiple paren-
chymal hemorrhages and KS-like lesions, which predomi-nantly developed around the pre-existing vascular chan-nels (Figure 1 1).
Nervous System
Kaposi's sarcomalike lesions were detected in several in-fected mice. These lesions apparently develop from thepia-arachnoidea of the brain and spinal cord. One animalhad an intracranial lesion adjacent to the trigeminal gan-
glia, while another animal had a similar growth within thelumbar nerve roots as these roots exit the spinal cord.These growths were moderately infiltrated by neutrophils,lymphocytes, and some macrophages. A meningeal le-sion composed of spindlelike cells and interspersed withirregularly shaped vascular channels lined by normal en-
dothelial cells is shown in Figure 12.
Lung
942 Stoica, Hoffman, and YuenAJPApril 1990, Vol. 136, No. 4
Figure 11. A KS-like lesion in the lung ofant~~~~~~~~.J
- elaWSt;wS f.,q4*'-w infaetctdedrmiouasre. ThaespindleUkieecrellespsrollif-ISS 5a erated primarily around the pre-existing?44; ~~~~~~~~vascular channels (V). H&E X260.
Figure 12. Cross section through the cervical spinal cord showing a segmental meningeal angiomatous KS-like lesion (arrow). Insetshows irregularly shaped vascular channels lined by normal endothelial cells. These vascular channels are surrounded by spindle-like cells. H&EX 60; inset x260. Figure 13. Transmission electron micrograph illustrating a vascular channel lined byplump endothe-lial cells (E) and an intraluminal erythrocyte (R). Several mature type C viral particles are seen within cytoplasmic spaces andwithin the lumen ofthe vascular channel (arrow). Insertshows a higher magnification ofthe type Cparticles (arrowhead). X43, 000;inset X60, 000.
Figure 14. Transmission electron micrograph showing aphagocytized erythrocyte (solid arrow) and a laminated body (arrowhead)in the cytoplasm ofa neoplastic cell. X40,000. Figure 15. Endotbelial (1) and spindlelike cells (2) ofa KS-like lesion when stainedwith UEA- 1 lectin were moderately labeled. H&E X 480.
Electron Microscopy
Electron microscopy of KS-like lesions showed the pres-ence of numerous mature C-type particles within intracel-lular and extracellular spaces of endothelial and spindle-like cells (Figure 13). In addition, erythrophagocytosis byendothelial and spindlelike cells were frequently observed(Figure 14). Evidence of neovascularization was demon-strated by the presence of primitive vascular spaces linedby spindlelike, normal, or plump endothelial cells and con-taining a few erythrocytes. Weibel-Palade bodies werenot detected by electron microscopy.
Lectin
The intensity of the nuclei and membranes of normal orplump endothelial cells, and surrounding spindlelike cellsof the KS-like nodules, when stained with FITC-labeledUEA-1 lectin, ranged from moderate to intense (Figure 15).
Histopathology Induced by MPSV
Less than 48-hour-old Balb/c mice infected intraperitone-ally with MPSV rescued by MoMuLV, and killed at 35 to 45
days after inoculation, manifested solid tumors in multipleorgans. The distribution of lesions was primarily within thespleen, skin, thigh muscles, lung, gastrointestinal tract,and liver. The most prominent lesions were found withinthe spleen, which were 2 to 5 times larger than normaland contained white foci of various sizes. These were his-tologically identified as fibrosarcomas (Figures 16, 17).The normal architecture of the spleen was completelyeffaced by proliferation of erythromyeloid cells, which alsoinfiltrated the fibrosarcomas, forming aggregates and is-lets of cells.
The thigh muscles were extensively infiltrated by fibro-sarcomas. The skin has subepidermal, multiple miliary fi-bromatous nodules, especially on the dorsal aspect of thebody. Histologic examination of the liver showed markedperiportal and sinusoidal myeloid infiltration. No concurrentKS-like lesions were observed in any of the MPSV-inocu-lated mice examined. Myeloproliferative sarcoma virus- in-duced fibrosarcomas did not stain with UEA-1.
DiscussionWe have demonstrated that MuSV349, in association withhelper virus, induced angiomatous lesions when injectedintraperitoneally into <48-hour-old Balb/c mice.
944 Stoica, Hoffman, and YuenAJPApril 1990, Vol. 136, No. 4
ww1116 K,~ N 17Figure 16. Spleen section ofa Balb/c mouse infected withMPSVvirus rescued withMoMuL V, showing afibrosarcoma (N) and effacedparenchymal architecture. H&E X 180. Figure 17. Higher magnification of area (N) of Figure 16 illustrating the proliferation offibroblastlike cells (arrow) admixed with myeloid cells (arrowhead). Note the absence ofvascular channels. H&E X 610
Each MuSV349-induced lesion is composed of irregu-larly shaped vascular channels lined by plump or normalendothelial cells, slitlike spaces filled with erythrocytes,and proliferating spindlelike cells. The tumor interspaceswere always infiltrated by neutrophils, lymphocytes, anda few macrophages. Some tumors were also infiltrated bynumerous eosinophils. Frequently, erythrophagocytosisby the plump endothelial or spindlelike cells was ob-served. Intracytoplasmic and interstitial hyaline bodiesalso were frequently seen in every infected tissue exam-ined. In addition, there were areas within the tumors thatresembled granulation tissue, which is probably a hostresponse to the lesions. We did not observe any regres-sion or disappearance of tumors during the lifespan ofinfected mice. The infected mice were very wasted whenmoribund.
The induction of angiomatous lesions has been re-ported in mice inoculated with Gz-MSV,8 or Balb MSVeand in rats inoculated with MPSV,27 or Harvey MSV.3031Although the angiomatous lesions induced by theseMuSVs have not been described in as great a detail as in
this report, they appear to share common features, suchas the presence of spindlelike cells, irregularly shapedvascular channels often lined by atypical endothelial cells,and an inflammatory component. Interestingly, like the an-
giomatous lesions induced by Ha-MSV,3' the MoMuSV-induced angiomatous lesions also closely resemble KS inhumans.
The characteristics that the MuSV349-induced KS-likelesions have in common with the more advancedKaposi's sarcoma are 1) proliferation of vascular channelslined with normal or atypical endothelial cells with largeovoid nuclei, 2) proliferation of spindlelike, somewhatpleomorphic cells with prominent ovoid nuclei, 3) the pres-ence of erythrocytes in slitlike spaces not lined by endo-thelial cells, and 4) erythrophagocytosis by endothelialand spindlelike cells and intra/extracellular accumulationof hyaline bodies.33"5 36Human KS and the MuSV349-in-duced KS-like lesions differ in the type of inflammatorycells present in the tumor. In KS, the primary componentis plasma cells, whereas in MuSV349-induced tumors,neutrophils are the main inflammatory cell type.
Beckstead and coworkers37 reported that, in KS, vascularspaces are sometimes lined with plump endothelial cells,as was observed in MuSV349-induced lesions. However,the formation of multilayers of endothelial cells lining vas-cular channels within the angiomatous lesions and the oc-casional formation of papillae by proliferating endothelialcells have not been reported in KS.
The histogenesis of KS and the KS-like lesions in-duced by the MuSVs remain uncertain. It is generally be-lieved that the cell of origin of KS is a multipotential mesen-chymal cell that gives rise to both the vascular and spindlecell components of the tumor.' A recent report incrimi-nated dermal dendrocytes in the histogenesis of KS.'Our studies on the MuSV349-induced KS-like lesions sug-gest that the spindlelike cells are less differentiated endo-thelial cells. Preliminary information derived from a timepoint experiment showed that the earliest MuSV349-in-duced lesions (14 days after inoculation) are sarcoma-touslike and are composed primarily of spindlelike cellsthat form slitlike spaces containing red blood cells (Yuenand Stoica: Unpublished observation). At approximately20 days after inoculation, clear angiomatous lesions de-velop in association with spindlelike cell proliferation. Thefirst lesions appear around the main abdominal vascularchannels, which suggests to us that virus circulating inthe blood infected perivascular stem cells and inducedthese cells to transform. We speculate that these trans-formed perivascular stem cells are the spindlelike cellsand it is these spindlelike cells that gave rise to the plumpendothelial cells lining the capillaries. This hypothesis ap-pears to be supported by the observation that both endo-thelial and spindlelike cells from the MuSV349-induced tu-mors bind UEA-1 lectin, which is considered a marker forhuman endothelial cells,' in contrast to the lack of UEA-1 binding to MPSV-induced tumors. Against this interpre-tation is the finding that UEA-1 does not bind to endothe-lial cells of mouse brain microvessels.i However, al-though we have confirmed this finding, we also haveobserved some binding of UEA-1 to the capillary endothe-lia in uninfected lung, spleen muscle, and stomach. It ap-pears, therefore, that endothelial cells from different or-gans and different species may differ in the sugar se-quences present on their membranes. The moderate tointense UEA-1 staining observed in the capillary endothe-lia of MuSV349-induced angiomatous KS-like lesionscould be due to the presence of UEA-1 - binding-specificsugar sequence in certain mouse endothelial cells. Itcould be argued that the binding of UEA-1 to the spindle-like cell component of the tumor could be due to the pres-ence of UEA-1 -specific sugar sequences of the virus en-velope glycoproteins. To distinguish these possibilities,additional studies on the binding of MoMuSV349 virionsto various lectins, and studies comparing the bindingcharacteristics of various lectins with MuSV349-induced
angiomatous lesions and MPSV-induced fibromatous le-sions from different organs with uninfected tissues of thecorresponding organs, are needed. Other studies that willbe helpful in determining histogenesis of these KS-like le-sions are searching for the presence of Weibel-Paladebodies and factor VIII, which are considered specific en-dothelial cell markers in MuSV349-induced KS-like le-sions. Regardless of the origin of the spindlelike cells, itappears that the vascular endothelia of MuSV349-in-duced tumors may be transformed.
The observation that angiomatous lesions are also in-duced by Harvey MSV,'3"3 which carries the ras onco-gene, is interesting. Because both v-ras and v-mos.arekinases, it suggests that the induction of KS-like lesions,in particular the transformation of endothelial cells, maynot be dependent on the expression of ras or mos, butrather on the overexpression of a kinase. In addition, be-cause MPSV induces fibrosarcomas in mice and angio-sarcomas in rats, it suggests that host factors may play arole in endothelial cell transformation.
Among the v-mos-containing murine sarcoma vi-ruses, MPSV infection in association with F-MuLV or Mo-MuLV in adult DBA/2 mice has been most extensivelystudied in vivo because of its unique ability to induce sple-nomegaly as a result of the proliferation of late erythroidprecursor cells, hematopoietic stem cells, and granulo-cyte-macrophage precursor cells,7 25'6 in addition to theinduction of sarcomas. These sarcomas are reported tobe composed of undifferentiated blast cells, fibroblasts,and granulocytic cells.7 Thus, the pathology induced byMPSV is in marked contrast to that induced by MuSV349and helper virus. Splenomegaly was not observed inMuSV349-infected newborn Balb/c mice. The mild my-eloid hyperplasia and neutrophilia observed in MuSV349-infected mice is not indicative of a myeloproliferative dis-ease, but is probably a response to the presence of solidtumors throughout the body and persistent viremia. In ad-dition, the preponderant proliferative cell type inMuSV349-induced KS-like lesions appears to be endothe-lial cells. To verify that the differences observed betweenMPSV and MuSV349 infections were due to differencesin the genetics of the viruses studied, we have obtainedMPSV-infected nonproducer cells by transfecting MPSVDNA into NIH/3T3 cells. The MPSV rescued with Mo-MuLV, when injected into newborn Balb/c mice, causedspleen enlargement (two-fold normal size) and inducedthe formation of small white miliary nodules. Histologic ex-amination showed that the normal splenic architecturewas completely effaced by an erythromyeloid cellular pro-liferation. The miliary white nodules were circumscribedareas of proliferating fibroblastlike cells. They were sur-rounded and infiltrated by aggregates and streams oferythroblastic and myeloblastic cells. Pre-existing vascu-lar channels observed in the nodules were lined with nor-
946 Stoica, Hoffman, and YuenAJPApril 1990, VoL 136, No. 4
mal endothelial cells. Within the thigh muscle, the nodulargrowths were characteristic of fibrosarcomas. Thus, thepathology induced by MPSV in association with MoMuLVin Balb/c neonates is in good agreement with that re-ported for MPSV-MuLV infections in adult DBA/2 mice.These findings clearly indicate that the pathology inducedby MuSV349-helper virus and MPSV-MoMuLV is dissim-ilar, and this difference may be attributable to differencesin the genetics of the viruses. The distinctive property ofMPSV is its ability to induce transformation of fibroblastsand erythromyeloid cells, in contrast to the ability ofMuSV349 to induce transformation of endothelial cells.The difference in the cell type transformed by these twoviruses provides a system to identify the viral genetic de-terminant crucial for disease specificity.
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