of such a cosmid insert should be a portionof the fragile site. (Microcell transfer of thetranslocations to fresh rodent backgroundsor isolation of the translocations throughchromosome sorting should significantlyenhance the ability to detect the transloca-tion junction.) Although it remains possiblethat the break actually occurred distal to thefragile site, the human portion of the trans-location junction would still be closer than 5cM from the site. Indeed, the genetic dis-tance of this region may be inflated becauseof unusually frequent recombination (19)such that a nearby DNA fragment may bephysically closer to the fragile site than thedistance of 5 cM suggests. This physicaldistance would approach the resolution ofpulsed field and field inversion gel electro-phoresis as well as "jumping" libraries (20).Furthermore, such nearby fragments, if notofthe fragile site itself, would be immediate-ly useful for linkage analysis in familiessegregating the fragile X chromosome (21).

Several conclusions can be drawn fromthe data presented. First, it is shown that afragile site can influence specific chromo-some breakage within the dividing cell. Sec-ond, this fragile site-mediated breakage canlead to nonrandom chromosome rearrange-ment involving the fragile site. This findingis of relevance concerning the postulatedrelationship between certain autosomal frag-ile sites and the breakpoints of nonrandomchromosome translocations seen in leuke-mias and lymphomas (5). Third, the obser-vation of reduced chromosome fragility atthe translocation junctions lends support forthe model of the fragile X site as a reiteratedDNA sequence. Lastly, the method of iden-tifying cell hybrids that carry chromosomerearrangements of a human fragile site androdent DNA suggests a means for molecularcloning offragile site sequences, and may beused for selected autosomal fragile siteswhere appropriate markers exist.

REFERENCES AND NOTES

1. G. R. Sutherland et al., Fragile Sites on HumanChrmosomes (Oxford Univ. Press, New York,1985).

2. G. R. Suthcrland, Trends Genat. 1, 108 (1985); Int.Rev. Cytol. 81, 107 (1983); R. L. Nussbaum and D.H. Lcdbetter,Annu. Rev. Genet. 20, 109 (1986).

3. S. L. Shermnan, N. E. Morton, P. A. Jacobs, G.Turner, Ann. Hum. Genet. 48, 21 (1984); S. L.Sherman at al., Hum. Genet. 69, 289 (1985).

4. G. Turner and P. A. Jacobs, Adv. Hum. Genet. 13,83 (1984).

5. J. J. Yunis, Science 221, 227 (1983); and A.L. Soreng, ibid. 226, 1199 (1984); M. M. LeBeauand J. D. Rowley, Nature (London) 308, 607(1984); F. Hecht and G. R. Sutherland, CancerGenet. Cytogenet. 12, 179 (179).

6. S. T. Warren and R. L. Davidson, Somat. CeUMol.Genet. 10, 409 (1984); R. L. Nussbaum, S. D.Airhart, D. H. Ledbetter, Hum. Genct. 64, 148(1983).

7. P. N. Nisen at al., N. Engl. J. Med. 315, 1139(1986).

8. V. A. McKusick, Mendelian Inheritance in Man

(Johns Hopkins Univ. Press, Baltimore, 1986); P.N. Goodfelow and K. E. Davies, Cytogenet. Cell.Genet. 40, 296 (1985).

9. A normal human male lymphoblastoid line (GM103-3A) was obtained from the Human GeneticMutant Cell Repository, Camden, NJ; a lympho-blastoid cell line established from a male with fragileX syndrome (MGL75) was obtained from P. A.Jacobs, Comell Medical School, and is described inP. A. Jacobs et al., Am. J. Hum. Genet. 34, 552(1982). The HGPRT/G6PD- Chinese hamster ova-ry cell line (YH21) was obtained from L. Chasin,Columbia, and is described in M. Rosenstraus andL. A. Chasin, Proc. Natl. Acad. Sci. USA. 72, 493(1975).

10. M. Siniscalco ct al., Proc. Nati. Acad. Sci. U.SA. 62,793 (1969).

11. M. Rosenstraus and L. A. Chasin, ibid. 72, 493(1975).

12. S. Dana and J. J. Wasmuth, Somatic Cell Genet. 8,245 (1982); C. H. C. M. Buys, G. H. Aanstoot, A.J. Nienhaus, Histochemistry 81, 465 (1984).

13. E. Southern, J. Mol. Biol. 98, 503 (1975); A. P.Feinberg and B. Vogelstein, Anal. Biochem. 137,266 (1984); G. M. Wahi, M. Stern, G. R. Stark,Proc. Natl. Acad. Sci. U.SA. 76, 3683 (1979); J.Meinkoth and G. Wahl, Anal. Biochem. 138, 267(1984).

14. I. Oberle ect al., Proc. Natl. Acad. Sci. U.SA. 83,1016 (1986); P. N. Goodfellow and K. E. Davies,Cytogenet. CeU. Genet. 40, 296 (1985).

15. M. H. Hofker et al., Am. J. Hum. Genet. 40, 312(1987).

16. U. Franke, B. Bakay, J. D. Connor, J. G. Coldwell,W. L. Nyhan, ibid. 26, 512 (1974).

17. D. H. Ledbetter, S. D. Airhart, R. L. Nussbaum,Am. J. Med. Genet. 23, 445 (1986).

18. D. H. Ledbetter, S. A. Ledbetter, R. L. Nussbaum,Nature (London) 324, 161 (1986).

19. P. Szabo et al., Proc. Natl. Acad. Sci. U.SA. 81,7855 (1984).

20. D. C. Schwartz and C. R. Cantor, Cell 37, 67(1984); G. F. Carle, M. Frank, M. V. Olson, Scince232, 65 (1986); F. S. Collins etal., ibid. 235, 1046(1987); A. Poustka and H. Lehrach, Trends Genet.2, 174 (1986).

21. S. T. Warren et al., Hum. Genet. 69, 44 (1985); W.T. Brown, A. C. Gross, C. B. Chan, E. C. Jenkins,ibid. 71, 11 (1985).

22. We thank R. L. Davidson for advice and supportduring the early portion of this work; T. W. Gloverand J. H. Priest for helpful discussion; P. Jacobs andL. Chasin for gifts of cell lines; and J. Gitschier, P.Jagadeeswaran, and P. L. Pearson for DNA probes;and L. Pearse for laboratory assistance. Supported,in part, by NCI grant CA31777 to R. L. Davidson,(University of Inois), NIH grant HD20521, aBasil O'Connor Starter Scholar Research Award(#5-600) from the March of Dimes Birth DefectsFoundation, and an Emory University WinshipCancer Center Grant to S.T.W.30 March 1987; accepted 8 May 1987

Smooth Muscle-Mediated Connective TissueRemodeling in Pulmonary Hypertension

ROBERT P. MECHAM, LOREN A. WHITEHOUSE, DAVID S. WRENN,WILLIAM C. PARKS, GAIL L. GRIFFIN, ROBERT M. SENIOR,EDMOND C. CROUCH, KURT R. STENMARK, NORBERT F. VOELKEL

Abnormal accumulation ofconnective tissue in blood vessels contributes to alterationsin vascular physiology associated with disease states such as hypertension andatherosclerosis. Elastin synthesis was studied in blood vessels from newborn calveswith severe pulmonary hypertension induced by alveolar hypoxia in order to investi-gate the cellular stimuli that elicit changes in pulmonary arterial connective tissueproduction. A two- to fourfold increase in elastin production was observed inpulmonary artery tissue and medial smooth muscle cells from hypertensive calves. Thisstimulation of elastin production was accompanied by a corresponding increase inelastin messenger RNA consistent with regulation at the transcriptional level. Condi-tioned serum harvested from cultures ofpulmonary artery smooth muscle cells isolatedfrom hypertensive animals contained one or more low molecular weight elastogenicfactors that stimulated the production ofelastin in both fibroblasts and smooth musclecells and altered the chemotactic responsiveness offibroblasts to elastin peptides. Theseresults suggest that connective tissue changes in the pulmonary vasculature in responseto pulmonary hypertension are orchestrated by the medial smooth muscle cell throughthe generation ofspecific differentiation factors that alter both the secretory phenotypeand responsive properties of surrounding cells.

P ERSISTENT PULMONARY HYPERTEN-

sion in infancy leads to severe lungvascular changes that include marked

hypertrophy, extension of vascular smoothmuscle to small vessels, adventitial thicken-ing, and increased connective tissue deposi-

R. P. Mecham, Respiratory and Critical Care Division,Department of Medicine, Jewish Hospital at Washing-ton Universit Medical Center, and Department of CellBiology and Physiology, Washington University Schoolof Medicine, St. Louis, MO 63110.L. A. Whitehouse, D. S. Wrenn, W. C. Parks, G. L.Griffin, R. M. Senior, Respiratory and Critical CareDivision, Department of Medicine, Jewish Hospital atWashington tUniversity Medical Center, St. Louis, MO

tion in both the media and adventitia (1, 2).In animal models of pulmonary hyperten-sion, increased collagen deposition appearsto be important in causing altered mechani-cal properties of hypertensive vessels (3, 4),but other connective tissue components also

63110.E. C. Crouch, Department of Pathology, Jewish Hospi-tal at Washington University Medical Center, St. Louis,MO 63110.K. R. Stenmark and N. F. Voelkel, Departrnents ofPediatrics and Medicine, Cardiovascular Pulmonary Re-search Laboratory, University of Colorado Health Sci-ences Center, Denver, CO 80262.

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Pulmonary artery Thoracic aorta

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Fig. 1. (A) Elastin production and elastin mRNAlevels in the lobar pulmonary artery and thoracicaorta of a control and hypoxic calf. Pulmonaryartery from the origin of the left middle lobe tothe sixth-order branch and a 3-cm segment of thethoracic aorta taken immediately distal to theductus were minced into approximately 1-mmcubes and incubated overnight at 37°C in high-glucose Dulbecco's modified Eagle's medium sup-plemented with nonessential amino acids, 30 mMHepes (pH 7.4), 10% fetal bovine serum, 1-aminopropionitrile (100 ,ug/ml), and penicilla-mine (50 ,ug/ml). Tropoelastin was extracted byhomogenizing in acetic acid (14) and assayed bycompetitive enzyme-linked immunosorbent assay(ELISA) (14). Values are expressed as nanogramsof a-elastin equivalents per microgram of DNA(mean ± SD of triplicate determinations) in 18hours. (Inset) Northern blot analysis of elastinmRNA levels in pulmonary artery. RNA was

A

isolated as described (15), and equal loadings (10,ug per lane) of glyoxyl-dimethyl sulfoxide-dena-tured total RNA were separated on a 1% agarosegel and blotted onto nitrocellulose. Hybridizationwas with nick-translated elastin probe pSS1 (16)and showed a single band at 3.5 kb. Left lane,control animal; right lane, hypoxic animal. (B)Elastin production by pulmonary artery medialSMCs from control and hypoxic animals. Conflu-ent first-passage cells, grown from explants ofarterial medial tissue, were incubated overnight inculture medium as described above. Tropoelastinin the medium and in a 0.5N acetic acid extract ofthe cell layer was determined by ELISA (14).Values are means ± SD of triplicate determina-tions of culture medium and cell layer combined.Total protein synthesis, as assessed by [3H]leucineincorporation into trichloroacetic acid-insolubleprotein, was similar for both control cells and cellsfrom hypoxic animals.

B

-F

5 -77

Pulmonary artery smooth muscle cells

increase in this condition. Elastin, the extra-cellular matrix protein responsible for elasticrecoil of arteries, is synthesized during vas-

cular development by smooth muscle cells(SMCs) (5). This highly cross-linked pro-

tein is localized principally in the mediallayer of the vessel, but in hypertensive pul-monary arteries, increased elastin can beobserved in the adventitia (2, 6, 7), suggest-ing that adventitial fibroblasts have under-gone a phenotypic transition to becomeelastogenic cells. This observation led us toutilize alterations in elastin synthesis as an

assay to investigate the molecular events andcellular signals responsible for differentialregulation of connective tissue productionin vascular disease.Pulmonary hypertension in the newborn

calf is an attractive model for studyingmechanisms underlying alterations in extra-cellular matrix accumulation associated withvascular abnormalities. The newbom calf,when placed at high altitude, develops hy-poxic pulmonary vasoconstriction and se-

vere pulmonary hypertension accompaniedby changes in lung vessels that mimic thosefound in infantile persistent pulmonary hy-pertension (7, 8). Indeed, after 2 weeks at a

simulated altitude of4300 m (440 mmHg),pulmonary arterial pressure is higher thansystemic pressure, and the pulmonary arteri-al vasculature shows medial and adventitialthickening, distal muscularization of thosepulmonary arterioles associated with de-

424

c

Fig. 2. Immunoperoxidase staining of pulmonary arteries with antibodies to elastin and type IVcollagen. Bovine lung tissues were fixed by perfusion through the airways with 10% neutral-bufferedformaldehyde solution and embedded in paraffin. For immunohistochemistry, 5-,um sections were

prepared as described (15) and incubated in a moist chamber for 2 hours at 37C with affinity-purifiedrabbit antibody to human type IV collagen (17) (10 mg/ml) or mouse monoclonal antibody[immunoglobulin G (IgG)] to bovine a-elastin (18) (25 mg/mi). Negative controls consisted of parallelsections incubated with normal mouse or rabbit IgG, respectively. Washed sections were incubated for20 minutes with affinity-purified biotin-conjugated goat antibody to mouse (BRL; 1:1600 inphosphate-buffered saline containing 1% bovine serum albumin) or goat antibody to rabbit IgG (BRL;1:800), washed, and incubated for 20 minutes with a 1:400 dilution of streptavidin-horseradishperoxidase (1:10). Complexes were visualized by incubation for 20 minutes with 3,3'-diaminobenzi-dine (Sigma; 0.5 mg/ml in 50 mM tris-HCI,pH 7.4) and H202. Sections were washed, counterstainedwith Harris hematoxylin, dehydrated, mounted in Permount, and examined by light microscopy.Original magnification, x 125. (A) Control animal. (B) Hypoxic animal. (C and D) Slightly larger,more proximal vessels from the same hypoxic animal as (B). (A) and (C) were stained with antibody toelastin and (D) was stained with antibody to type IV collagen. The thickened and hypercellularadventitia in hypertensive vessels contains numerous fibers which stain for elastin (B and C) whereastype IV staining surrounds individual SMCs in the thickened media (D).

creased luminal diameter, and an eventual freed the left middle lobe pulmonary arteryloss ofvasoreactivity due in part to increased from adherent lung tissue through the sixth-vascular connective tissue (4). order branch. When elastin production was

To compare elastin production in vessels determined in the minced vessel, we foundofcontrol animals and animals that had been elastin synthesis to be approximately fourmade hypoxic by simulated high altitude, we times higher in the pulmonary artery from

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U)

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U-c

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15

10

UnconditionedTA-hypoxic

LPA-controI

LPA-hypoxicLPA-hypoxic (IV)

T

Tr

T

MPA-Adv FCL-1 20 FCL-270

Fig. 3. The effect of smooth muscle-conditioned serum on elastin production by fibroblasts. Shfrom medial tissue explants of thoracic aorta (TA) and lobar pulmonary artery (LPA) from a hypand control calfwere seeded into 900-cm2 roller bottles and maintained in growth medium consisiof Dulbecco's modified Eagle's medium supplemented with 20 mM Hepes, nonessential amino acand 10% fetal bovine serum. Shortly after the cells reached visual confluency, the growth mediumreplaced with 100% calf serum for 18 hours. The conditioned serum was then diluted to 10% v

growth medium or was fractionated by Sephadex G-100 chromatography; the fraction contairelastogenic activity (fraction IV) was lyophilized, diluted with Dulbecco's medium to the volumserum applied to the column, and diluted to 10% with growth medium. Freshly passaged fibrobifrom the adventitial layer of main pulmonary artery of control animals (MPA-Adv) or fromligamentum nuchae ofeither 120-day (FCL-120) or 270-day (FCL-270) fetal calves were then cultbin growth medium supplemented with conditioned serum or fraction IV. Unconditioned calf setdiluted in growth medium or fractionated over Sephadex G-100 served as control. After confluencyreached, elastin production was determined for each cell type by competitive ELISA (14). Valuesmeans and standard deviation of triplicate determinations.

hypoxic animals than that from controls(Fig. IA). Northern blot analysis of RNAextracted from the tissue and hybridizedwith a DNA probe for elastin demonstratedan increase in elastin-specific messengerRNA (mRNA), thereby correlating the in-crease in elastin synthesis with increasedmRNA levels. In contrast to the differencesobserved in the pulmonary artery, elastinproduction and elastin mRNA levels in thethoracic aorta from control and hypoxicanimals were not significantly different (Fig.LA). That the thoracic aorta was unaffectedin the animals made hypoxic by simulatedhigh altitude was not surprising since blood

Fig. 4. Induction of chemotactic responsivenessto elastin peptides. Ligamentum nuchae fibro-blasts from a 95-day bovine fetus were cultured ingrowth medium alone (control), in growth medi-um containing the Sephadex G-100 fraction ofsmooth muscle-conditioned serum that con-tained elastogenic activity (conditioned IV) (seelegend to Fig. 3), or the corresponding SephadexG-100 fraction of unconditioned serum (uncon-ditioned IV). Chemotactic responsiveness to elas-tin peptides and to 1 nM PDGF (30 ng/rnl) wasassayed by a double micropore membrane systemin modified Boyden chambers as described (19).Values are expressed as net cells per high powerfield and are means ± SE for 15 determinations.

pressure in the systemic circulation isincreased at high altitude (7).The results described above demonstrai

dramatic increase in the rate of elastin sthesis in the hypertensive pulmonary artuTo determine whether these changes resied from stable phenotypic alterationsvascular cells, we compared elastin prod

*-- Control-. Unconditioned fraction B

40 - 0O Conditioned fraction IV

Co

20

0S 30

' 100E_

1

tion in cultured pulmonary artery medialSMCs from control and hypoxic animals.Cultured SMCs from hypoxic animals pro-duced more elastin than control SMCs (Fig.1B). The increase in elastin production ap-pears to reflect a stable phenotype sincerelative differences between control cells andcells from hypoxic animals persisted in vitrothrough at least three culture passages.

Histochemical studies of smaller elasticand muscular arteries in the hypertensive ratlung have revealed extensive remodeling inthe adventitia as well as in the media (6, 7).In particular, a marked accumulation ofelastin-rich matrix external to the mediaindicates increased elastin synthesis by theadventitial cells. A similar increase in elastin-rich matrix in hypertensive bovine lung wasconfirmed by immunoperoxidase stainingwith antibodies to elastin. Elastin staining

4Cs was observed in all layers of the artery butwas markedly increased in the adventitia

-ig (Fig. 2). Staining with monospecific anti-was bodies to type IV collagen, which stains thevith basal lamina ofSMCs, fiuther demonstrateding the marked increase in connective tissuee of external to the thickened media.laststhe Little is known about how the adventitialired cell might be modulated to express therum elastin phenotype. Our previous finding thatwas SMCs can influence elastin synthesis by

cultured endothelial cells (9) suggested thepossibility that the elastin phenotype is in-fluenced in the diseased vessel by specific

not factors released locally by activated SMCs.Early-passage medial cells in roller bottles

te a were exposed overnight to 100% calf serumyn- to test for the presence of an elastogeniccry. differentiation factor released by the SMCsult- from hypoxic animals. Samples ofthe condi-in tioned serum were then tested for effects on

Luc- elastin synthesis in fibroblasts isolated from

oc_2 o_- _

c C)o0-

0 1.0 5.0 10.0Elastin peptides (gg/mi)

/

PDGF (30 ng/ml)

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adventitia of normal pulmonary artery. Pro-duction of elastin by the adventitial cell wasenhanced by serum conditioned by SMCsfrom the pulmonary artery of hypoxic ani-mals, but not by unconditioned serum or byconditioned serum from aortic cells or frompulmonary vascular cells of control animals(Fig. 3). The elastogenic stimulatory effectseems to be a response of the SMC to highserum levels since activity could not bedetected in culture medium supplementedwith only 10% (v/v) calfserum. The amountof stimulatory activity present in condi-tioned serum showed variation with differ-ent serum lots, and consistently greater ac-tivity was obtained with calf serum thanwith fetal bovine serum.To determine whether the smooth mus-

de-derived elastogenic factor (SMEF) actsat a transcriptional or post-transcriptionallevel, we studied the effects of conditionedserum on the differentiation offetal ligamen-tum nuchae fibroblasts. In these cells, theelastin phenotype is actively expressed in lategestation (older than 180 days), whereas inearly gestation the phenotype is inactive butinducible (10). Thus, ligament fibroblastsearly and late in gestation can be used in aconvenient well-characterized bioassay todetermine whether factors induce new elas-tin gene expression or simply stimulate elas-tin synthesis in cells that are already activelyexpressing the elastin phenotype.When differentiated (late gestation) liga-

ment fibroblasts were cultured in mediumcontaining serum conditioned by SMCsfrom the pulmonary artery of hypoxic ani-mals, there was a two- to fourfold stimula-tion of elastin production compared to cellsgrown in unconditioned medium or in se-rum conditioned by SMCs from normalpulmonary artery or thoracic aorta (Fig. 3).Total protein synthesis, as determined by[3H]leucine incorporation into trichloroace-tic acid-insoluble protein, was unaltered bythe conditioned medium. Significantly, un-differentiated (early gestation) cells culturedin the presence of conditioned serum fromthe SMCs of hypoxic animals took on phe-notypic and morphological features of dif-ferentiated cells, as evidenced by the onset ofelastin production (Fig. 3) and a stellatemorphology. These results demonstrate thatSMEF functions both as an enhancer ofelastin synthesis and as a differentiation fac-tor, inducing expression of the elastin phe-notype in responsive cells.The inductive property of smooth mus-

cle-conditioned serum was substantiated byinvestigating its effects on another differen-tiation-related property of ligament cells-namely, chemotaxis in response to elastin-

derived peptides. We previously showedthat the appearance of chemotactic respon-siveness to elastin peptides in fetal ligamentfibroblasts parallels the onset of elastin syn-thesis and that both of these phenomena aresensitive markers of differentiation (11).Figure 4 shows that undifferentiated liga-ment cells grown in medium supplementedwith column fraction IV from SMC-condi-tioned serum (see below) acquired chemo-tactic responsiveness to elastin peptidescharacteristic of differentiated cells. No che-motactic response was observed when cellswere grown in fraction IV from uncondi-tioned serum, even though the cells re-mained fully responsive to the fibroblastchemoattractant platelet-derived growth fac-tor (PDGF). These results not only provideevidence for an altered secretory phenotypein response to SMEF, but also suggest thatcell surface receptors are changing as well.Cell surface modulation could have impor-tant physiological consequences by alteringreceptor-mediated responses such as cellmovement or the ability of the cell to re-spond metabolically to external stimuli.Thus, both the secretory and responsiveproperties of the cell may be changed afterexposure to SMEF.The biochemical characteristics of the

elastogenic activity released by hypertensivepulmonary artery SMCs are not yet known.Indeed, it is not dear whether SMCs pro-duce SMEF directly or whether they processor alter serum or plasma factors to produceSMEF. It is also uncertain whether theactivity involves more than one factor.When SMEF-containing serum was frac-tionated by Sephadex G-100 chromatogra-phy, elastin-stimulating activity was foundas a broad peak eluting near the columntotal volume (fraction IV), correspondingto a molecular weight range of less than15,000 (based on the exclusion properties ofglobular proteins). Cells grown in mediumcontaining this fraction showed changesidentical to those described above for cellsgrown in the presence of unfractionatedconditioned serum (Figs. 3 and 4).

In summary, these results suggest that thepulmonary artery SMC plays a critical rolein the pathogenesis of vascular changes ofpulmonary hypertension by modifyfing thephenotype of surrounding cells in the vesselwall. Our data support a model in whichexposure of medial SMCs to plasma factorsafter vessel wall injury results in a phenotyp-ically altered cell that generates SMEF.SMEF, in turn, stimulates or induces elastinsynthesis and perhaps synthesis of otherconnective tissue macromolecules. Also, be-cause SMEF or an associated activity affects

chemotactic responsiveness ofvascular cells,smooth muscle factors could modulate re-sponsiveness to external stimuli generally. Ifthe SMC assumes an autocrine-like functionin the hypertensive state after vessel injury,these findings could have significance forother vascular diseases such as atherosclero-sis, in which there is evidence that the SMC(12) is responsible for many of the cellularand functional features ofthe atheroscleroticlesion (13). The finding that the pulmonaryartery SMC in pulmonary hypertension gen-erates a factor (or factors) that affects con-nective tissue synthesis by other cells in thevessel wall provides a direction for fiurtherresearch into molecular and cellular mecha-nisms of connective tissue regulation in dis-eased vessels.

REFERENCES AND NOTES

1. S. D. Haworth and L. Reid, J. Pedier. 88, 614(1976); S. J. Soifer and M. A. Heymann, C!in.Perinat. 3, 745 (1984); R. L. Geggel and L. M.Reid, ibid., p. 525.

2. B. Meyrick and L. Reid, Am. J. Pathol. 100, 151(1980); J. D. Murphy, M. Rabinovitch, J. D.Goldstein, L. M. Rcid,J. Pedatr. 98, 962 (1981).

3. H. Wolinsky, Circ. Re. 26, 507 (1970).4. J. K. Kcrr, D. J. Rilcy, M. M. Frank, R. L. Trestad,

H. M. Frankel,J. Appl. Phsic!. 57, 1760 (1984).5. S. M. Partridge,Ad. Protein Chem. 17, 227 (1962);

J. M. Glark and S. Glagov, Arteriosderosis 5, 19(1985); B. Faris etal.,Biocbim. Biophys.Acta 797, 71(1976); F. W. Keelcy, Can. J. iochaem. 57, 1273(1979).

6. B. Meyrick and L. Reid, Lab. Invest. 38, 188(1978); M. Rabinovitch, W. Gamble, A. S. Nadas,0. S. Miettinen, L. Reid,Am.J. Physic!. 236, H818(1979); B. Meyrick and L. Reid, Ecp. Lung. Ra. 2,257 (1981); L. Rcid, Chest 89, 279 (1986).

7. K. Stenrnark at al.,J. Appl. PAysic!. 62, 821 (1987).8. J. T. Reeves and J. E. Leathers,Anat. Rec. 157, 641

(1967); B. F. Grover, J. T. Reeves, D. H. Will, S.G. Blount,J. AppI. Physiol. 18, 567 (1963).

9. R. P. Mecham, J. Madaras, J. A. McDonald, U.Ryan, J. Cel. Physic!. 116, 282 (1983).

10. B P. Mecham, J. Madaras, R. M. Senior, J. CeUBid. 98, 1804 (1984); R. P. Mecham, S. L. Morris,B. D. Levy, D. S. Wrenn,J. Bid. Chem. 259, 12414(1984).

11. R. P. Mecham, G. Griffin, J. Madaras, R. M. Senior,J. Cell Bid. 98, 1813 (1984).

12. J. C. Geer, H. C. McGill, J. C. Strong, Am. J.Pathol. 38, 263 (1961); J. Chamely-Campbell, G.R. Campbell, R. Ross, Phyil. Rev. 59,1 (1979); J.Grunwald and C. C. Haudenschild, Artenicsdrosis4, 183 (1984).

13. R. Ross and J. A. Glomset, Scienc 180, 1332(1973); N. Engl. J. Med. 295, 369 (1976); K.Pietila and T. Nikari, Med. Bid. 61, 31 (1983); R.Ross, N. Eng!. J. Med. 314, 488 (1986).

14. D. S. Wrenn, G. L. Griffin, R. M. Senior, R. P.Mecham, Biochemistvy 25, 5172 (1986).

15. D. S. Wrenn etal.,J. Biol. Chem. 262, 2244 (1987).16. K. Yoon at al., Arch. Bichem. Bwphys. 241, 684

(1985); G. Giro at al., J. C!in. Invest. 75, 672(1985).

17. E. Crouch, F. Quinones, D. Chang, Am. Rev.Repir. Dis. 133, 618 (1986).

18. R. P. Mecham and G. Lange, Biochemsrtry 21, 669(1982).

19. R. M. Senior, G. L. Griffin, R. P. Mecham,J. Clin.Invest. 70, 614 (1982).

20. We thank J. Davidson for providing the elastin-specific probe pSS1 and 1 Reeves, J. Madaras, B.Levy, M. Seeman, D. Cox, J. Fasules, and E. C.Orton for assistance and discussion. Supported byU.S. Public Health Service grants HL-26499, HL-29594, HL-14985 and clinical investigator awardHL-01503 (to K.R.S.).6 February 1987; accepted 20 May 1987

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