Hypertensive Pulmonary Arteries - Europe PMC

Amerncan Journal of Pathology, Vol. 140, No. 2, February 1992 Copyright X) American Association of Pathologizsts

Extracellular Matrix Protein Gene Expression in Atherosclerotic Hypertensive Pulmonary Arteries Mitchell D. Botney,* Larry R. Kaiser,t Joel D. Cooper,t Robert P. Mecham,*t Deena Parghi,* Jill Roby,* and William C. Parks* From the Departments of Medicine,* Surgery, t and Cell Biology,* Jewish Hospital, Washington University Medical

Center, St. Louis, Missouri

Lobar pulmonary arteries from patients with unexplained pulmonary hypertension were obtained at the time of single-lung transplantation to determine the response of large elastic vessels to increased intraluminalpressure. Specifically, human pulmonary arteries were examined to determine if remodeling remained active at the time of surgery and whether remodeling was similar to previously reported remodeling observed in several animal models. Grossly, the hypertensive vessels appeared atherosclerotic Histochemical stains revealed a thick, diffuse neointima in hypertensive vessels compared with normal vessels. Immunohistochemistry demonstrated elastin protein in the neointima and in situ hybridization studies demonstrated tropoelastin mRNA largely in the neointima Similarly, immunohistochemistry and in situ hybridization detected cellularfibronectin, thrombospondin and type I collagen protein and mnRNA within the thickened intima from hypertensive vessels. These studies provide evidence that hypertensive vessels in patients with severe chronic pulmonary hypertension are actively remodeling but that the pattern of remodeling is different from previously described animal models. (Am J Pathol 1992, 140:357-364)

Human pulmonary hypertension develops in response to a large variety of diseases.1 The nature of the response appears to differ depending on the age at which pulmonary hypertension develops and duration of disease. When pulmonary hypertension is present at birth but survival is short, for example, in neonates with persistent fetal circulation, remodeling is accompanied by medial hypertrophy.2 When pulmonary hypertension is present at birth

but survival is prolonged or in older individuals developing pulmonary hypertension after birth neointimal hyperplasia and atherosclerotic lesions are commonly observed.3 Histologic studies of human hypertensive pulmonary arteries suggest vascular remodeling is a common abnormality regardless of etiology.2 Generally, vascular remodeling in the pulmonary microcirculation is believed to dispose to increased pulmonary artery pressure while remodeling in the large elastic vessels is an adaptive response to increased pressure. The specific mechanisms, however, underlying both large and small vessel remodeling in human pulmonary hypertension remain largely unknown. We have had the opportunity to study adult human lobar pulmonary arteries obtained at the time of singlelung transplantation for severe, unexplained pulmonary hypertension. These vessels were examined to determine 1) if active remodeling was still present; and 2) whether remodeling in human pulmonary hypertension was similar to remodeling observed in animals. Our findings suggest these vessels are actively remodeling despite the chronicity of disease but that the pattern of remodeling, at least at the time of surgery, is different from most animal models of pulmonary hypertension.

Methods Tissue Lobar pulmonary arteries were obtained from the excised lungs of patients undergoing single lung transplant surgery for unexplained pulmonary hypertension at Washington University Medical Center. Normal pulmonary arSupported by grants HL-29594, HL-02425, and HL-41040 from the National Institutes of Health and by a Grant-in-Aid from the American Heart Association. Mitchell D. Botney is a recipient of a Physician-Scientist Award from the National Institutes of Heafth. Accepted for publication August 28, 1991. Address reprint requests to Dr. Mitchell Botney, Respiratory and Critical Care Division, Jewish Hospital, 216 S. Kingshighway Blvd., St. Louis, MO 6311 0.

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teries were obtained from lung transplant donors. Clinical features of patients and donors are summarized in Table 1. Immediately following surgical resection, pulmonary arteries from transplant recipients were processed for histological study and in situ hybridization. Lungs from normal donors were treated prior to harvest to minimize ischemic injury: a single bolus of prostaglandin E1 (500p,g) followed by 3 L modified Eurocollins solution (10 mEq/L Na+, 108 mEq/L K+, 14 mEq/L C1 -, 12 mEq/L Mg+2, 12 mEq/L So4-2, 85 mEq/L HPO4 2,15 mEq/L H2PO4-, 3.27% glucose, 40C) was rapidly administered via the pulmonary artery. Donor lungs were maintained at 40C in normal saline until processed for histological study and in situ hybridization. Pulmonary arteries from transplant recipients and normal donors were otherwise treated identically.

Immunohistochemistry Lobar pulmonary artery segments were fixed in 4% buffered formaldehyde for 6 hr at room temperature and subsequently dehydrated in sequential 30%, 50% and 70% ethanol washes. Tissues were embedded in paraffin and prepared for immunoperoxidase staining as described previously.4 Endogenous peroxidase was blocked with 0.3% (v/v) H202 in methanol for 20 minutes at room temperature. Nonspecific immunoglobulin binding sites were blocked with normal goat serum. Sections were subsequently incubated for 1 hour at 370C with rabbit polyclonal antihuman elastin serum,5 mouse monoclonal anticellular (EDIII domain) fibronectin antibody,6 mouse monoclonal antithrombospondin antibody,7 mouse monoclonal antibody to the amino-terminal propeptide of human procollagen type 18 or mouse monoclonal antibody to human type IV collagen.9 Appropriate preimmune sera served as negative controls. Sections were then incubated for 20 minutes with affinity-purified biotinconjugated goat antirabbit or antimouse IgG (1 :1600 dilution), washed, and incubated for 20 minutes with horseradish peroxidase-streptavidin (1:400 dilution). Immunoglobulin complexes were then visualized by incubation with 3,3'-diaminobenzidine (0.5 mg/ml in 50 mM TrisHC1, pH 7.4), 3% H202 and NiCI2. Sections were

washed, counterstained with Harris hematoxylin, dehydrated, mounted in Permount, and examined by light microscopy.

In Situ Hybridization [35S]-labelled RNA probes were transcribed in vitro from human tropoelastin (HEL-2) (gift of Dr. J. Rosenbloom), human fibronectin (pFH-6) (gift of Dr. A. Kornblihtt) and human thrombospondin (TXE6S) (gift of Dr. W. Frazier) cDNA probes using t[35S]-UTP (> 1200 Ci/mmol, ICN Biochemicals, Irvine, CA)4 HEL-2 spans the region encoded by exons 7 through 15 of tropoelastin mRNA10 and pFH-6 is complementary to the first four type repeats of fibronectin mRNA. 1 TXE6S is a full-length cDNA complementary to the coding and 3'-noncoding region of thrombospondin mRNA.12 The length of each antisense probe was 600, 5800, and 870 nt, respectfully. The 5800 nt TXE6S cRNA was subsequently hydrolyzed by incubation in 0.2 M carbonate at 600C to an average fragment length of approximately 200 nt. [35S]-labeled T66, a 500 nt sense RNA probe transcribed from a bovine tropoelastin cDNA, served as a negative control. This RNA is 68% GC-rich and should have a propensity for nonspecific hybridization. Therefore, lack of in situ hybridization signal with this probe indicates appropriately stringent wash conditions. Sections of paraffin-embedded normal and hypertensive lobar pulmonary artery segments from patients and donors were incubated with [35S]-labeled HEL, pFH-6 or TXE6S antisense RNA probes as described.4 As controls, some sections were treated with 100 ,ug/ml RNase A (Sigma, St. Louis, MO) to remove endogenous RNA. Sections were pretreated with 1 ,ug/ml nuclease-free proteinase K (Sigma) and washed in 0.1 M triethanolamine buffer containing 0.25% acetic anhydride. Hybridization solution containing 2.5 x 105 cpm of [35S]-labeled probe was added to the processed sections and slides were incubated overnight at 55°C. After hybridization, slides were washed extensively under stringent conditions. To decrease background, slides were incubated with 20 ,ug/ml RNase A to remove unhy-

Table 1. Clinical Characteristics of Normal Donors (NL) and Patients with Unexplained Pulmonary Hypertension (PHTN) PAP mmHg Age Duration Patient Disease Gender (mos) (mean) (yr) 2 3 4 5

PHTN PHTN PHTN NL NL

36 32 32 35 28

F F F F M

57 58 60

9 24 6

Medications

PGI2 Diltiazem Diltiazem

Matrix Proteins in Human Pulmonary Hypertension

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bridized probe. Washed slides were then processed for autoradiography.

Results

Subjects Three patients with unexplained pulmonary hypertension were female, between the age of 30-40 years, and had mean pulmonary artery pressures between 55-60 mm Hg (Table 1). All were being treated with calcium-channel antagonists (nifedipine or diltiazem) or prostacyclin

(PGI2). The two normal donors, a 35-year-old female and a 28-year-old male, died of intracranial hemorrhage and head trauma, respectively. The patients did not have any underlying illness and were not taking medications.

Histology The histology of hypertensive pulmonary arteries differed dramatically from comparable normal large elastic arteries (Figure 1). The intima of normal main, lobar, and segmental pulmonary arteries was thin and uniform in thickness. The endothelial cells were closely apposed to the internal elastic lamina and little, if any, extracellular matrix was present in the subendothelial space. The thickness and configuration of elastic lamellae in the medial layer

A

appeared normal. The smooth muscle cells appeared normal in size and shape. The predominant change in the hypertensive vessels was a markedly thickened intima composed of cells and extracellular material sandwiched between the endothelial layer and internal elastic lamina (Figure 1 B). This neointima was not confined to focal lesions, but rather, was uniformly distributed throughout the large pulmonary vessels in all patients. The thickness of the neointima, however, did vary from patient to patient. No platelets were observed adherent to the endothelial layer. Obvious changes in the lamellar unit structure were not apparent, although mild medial hypertrophy was present, and the configuration of elastic lamellae in the medial layer as viewed by Verhoeff-van Giesen elastin stain was consistent with development of pulmonary hypertension in adult life.13 The presence of elastin in the neointima was suggested by the Verhoeff-van Gieson elastin stain and confirmed with a human elastin antibody5 which stained fibers in both media and neointima (Figure 2). Infrequently, two other patterns of vascular remodeling were noted in hypertensive vessels. There were occasional fibrous atherosclerotic plaques accompanied by underlying medial thinning. In other areas, medial fibromuscular hypertrophy was seen accompanied by less severely thickened intima (data not shown). Unfortunately, technical factors precluded fixation of pulmonary arteries at clinical pressures. Therefore, we were unable to quantify the dimensions of the arterial wall.

B

Figure 1. Normal and hypertensive pulmonary artery histology. Human lobarpulmonary arteriesffrom patients with pulmonary hypertension undergoing unilateral pulmonary transplantation were resected, fixed in 4% buffered formaldebyde, and stained with Verhoeff-van Gieson (VG) elastin stain. Marked neointimal hyperplasia is present in the hypertensive vessel (B) compared with the normal vesselfrom the same anatomic level (A). The tvpical lamellar structure is absent in the neointima (magnification, x 100).

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Figure 2. Immunohzstochemical localization of extracellular matrix components in the thickened intimza from hypertenzsive pulmonary Hypertensive pulmonary arteries were fixed in formalin and embedded in paraffin. Immunohistochemistiy were performed as described in Methods with antibodies for cellular fi'bronectin (B), thrombospondin (C), elastin (D), type IV collagen (E), and type I procollagen (F) Cellular fibronectin is present only within the thickened intima i) of the hypeensive vessel. No staining is observd in the medial layer (i) Thrombospondin is concentrated within endothelial cells and within scattered cells of the intima. Light staining appears within the media Elastin and type IV staining ispresent within both the intima and media. Stainingfor type Iprocollagen is lagely confined to the thickened intimza Appropriate normal serum controls were negative. H&E stain of hypertensive pulmonary artery (A). arteries


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Immunohistochemistry and In Situ Hybridization Previous in situ hybridization studies in the neonatal bovine calf model of hypoxic pulmonary hypertension demonstrated strong signals for tropoelastin mRNA in the medial layer of large pulmonary arteries.4 Vessels from normal calves had strong signals associated with cells in the inner medial layer, whereas vessels from hypoxic hypertensive calves had strong signals within the outer medial layer. To determine if a similar response was present in human vessels, in situ hybridization studies were performed with human pulmonary arteries. In contrast to vessels from hypertensive calves, positive signal for tropoelastin mRNA was detected predominantly in cells within the neointima as well as rare, scattered cells within the medial layer of hypertensive vessels (Figure 3). Similarly, only scattered cells in the medial layer from normotensive humans were positive for tropoelastin mRNA. Thus, chronically hypertensive adult human vessels differed markedly from neonatal bovine hypertensive vessels by the presence of marked neointimal hyperplasia associated with tropoelastin mRNA in the neointima but not in the media. The presence of additional extracellular matrix proteins was studied by immunohistochemical techniques to determine if the changes seen in the neointima were unique to elastin. Diffuse, intense staining was present in both the neointimal and medial layers from hypertensive vessels using an anti-type IV collagen antibody. A similar staining intensity was observed over the media from normal vessels (data not shown). The presence of fibronectin was also demonstrated with a specific anticellular fibronectin antibody. Staining was present within the hyperplastic intima from the hypertensive vessel (Figure 2B), whereas little staining was observed over the medial layer. There was also little staining for cellular fibronectin in the intima or media of normal pulmonary arteries (data not shown). Thus, in contrast to elastin and type IV collagen, cellular fibronectin protein was observed only in the thickened neointima from remodeling vessels and not the medial layer. Immunohistochemical studies were also performed with an anti-thrombospondin antibody. Weak staining of the intima and somewhat more intense staining along the endothelial layer from hypertensive vessels was observed (Figure 2C). Weak staining was also observed over the media from hypertensive vessels with slightly greater staining of the media observed in normal vessels compared with hypertensive vessels. To determine whether collagen synthesis was occurring, immunohistochemical staining for al(l) procollagen was performed with an antibody to the amino-terminal end of the procollagen propeptide. This antibody identi-

fies newly synthesized al(l) procollagen before cleavage of the amino-terminal end and does not recognize mature collagen fibers. A pattern of staining similar to cellular fibronectin was seen with the al(l) procollagen antibody (Figure 2F). Staining was intense over the area of hyperplastic intima from the hypertensive vessel with little staining observed over the medial layer from these vessels. There was no significant staining for type procollagen in normal pulmonary arteries (data not shown). In situ hybridization studies were performed with human pulmonary arteries to determine if cells in the neointima were expressing fibronectin, thrombospondin and type collagen mRNA in addition to tropoelastin mRNA. After 4D exposure, positive signal for fibronectin mRNA was observed mainly in cells scattered throughout the thickened intima of hypertensive pulmonary arteries (Figure 3B). Few positive signals were observed in medial cells, correlating with the immunohistochemical studies. Normal arteries also showed positive signals for fibronectin mRNA in occasional medial cells (Figure 3A). Thrombospondin mRNA, after 1 4D exposure, was seen both in cells throughout the intima of hypertensive vessels and in cells on the luminal edge of the intima in hypertensive vessels (Figure 3D). These luminal cells are probably endothelial cells rather than platelets since endothelial denudation with platelet adherence is not a feature of primary pulmonary hypertension. Few positive cells were observed in normal vessel media but positive cells (macrophages) were observed regularly in the adventitia (Figure 3C). Type collagen, after 5 weeks of exposure, was detected in intimal cells from hypertensive vessels in a pattern similar to fibronectin and tropoelastin (data not shown). In addition, some cells in the adventitia but not media were positive. No positive signals were observed in normal vessels.

Discussion Remodeling in adult large pulmonary vessels is considered an adaptive response to increased blood pressure irrespective of the etiology rather than a primary disorder.14 Unlike the systemic vasculature, neointimal hyperplasia and atherosclerosis are uncommon in pulmonary arteries. Although neointimal hyperplasia may be found in human systemic arteries at an early age3 13 the large elastic arteries of the pulmonary vasculature remain free of neointimal hyperplasia even in the elderly15 or in the presence of the usual risk factors such as diabetes, tobacco smoke, or hypercholesterolemia. The absence of neointimal hyperplasia probably reflects the substantially lower blood pressures present in pulmonary arteries.3 Thus, the presence of significant neointimal hyperplasia in the large elastic pulmonary arteries must be consid-

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Figure 3. In situ hybridizationforfibronectin, thrombospondin and tropoelastin mRNA Normal (A,C,E) and hypertensive (B,D,F) pulmonary arteries were hybridized in situ with antisense [-5S]-labeled cRNA probesforfibronectin (pFH-6), thrombospondin (7XE6S), or tropoelastin (HIEL-2) mRNA. Exposure times u'ere 3 days, 2 weeks, and 3 weeks, respectiveh' [-5S]-anti-T66, a sense bovine tropoelastin cRNA, served as a negative control. Cells positive forfibronectin mRNA were largely confined to the hbperplastic intima from the hypertensive pulmonary artery (B). Feun positive cells u'ere present in normal artery (A). Cells positive for thrombospondin mRNA were scattered throughout the thickened intima of the hypertensive vessel (D) and in cells lining the vessel lumen, suggesting endothelial cells were also expressing thrombospondin mRNA. No signal uwas present in normal media (C) but several macrophages in the adherent adventitialfascia of both normal and hbpertensive vessels were positive. Positive signals for tropoelastin mRNA u'ere detected predominantly in cells within the neointima as uwell as rare, scattered cells uithin the medial layer of hypertensive vessels (F) but no signal was observed in normal vessels (E). No signal was observed uith[-35S]-anti-T(6 cRNA or after RNase A digestion.


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ered abnormal and reflects a stereotyped vascular response to isolated high intraluminal pressure. Although little is known about the composition and origin of neointimal hyperplasia in hypertensive pulmonary arteries, these vessels appeared grossly atherosclerotic. Histochemical stains revealed a thick neointima in hypertensive vessels compared with normal vessels. Elastin was present in the neointima but appeared disorganized compared with the normal lamellar structure present in the media. In situ hybridization studies demonstrated active tropoelastin gene expression largely in the neointima. Similarly, immunohistochemical and in situ hybridization studies detected synthesis and deposition of other extracellular matrix components, such as cellular fibronectin and type collagen, within the thickened intima from hypertensive vessels. These studies provide evidence that hypertensive vessels in patients with severe chronic pulmonary hypertension are actively remodeling. These observations in human pulmonary hypertension present an interesting contrast with several experimental animal models of pulmonary hypertension. For example, marked medial hypertrophy without diffuse intimal thickening or atherosclerosis is a common feature of large vessel remodeling in animal models of hypoxic pulmonary hypertension.1-19 Increased elastin synthesis and deposition is observed in the medial layer of the large elastin vessels in several animal models of hypoxic pulmonary hypertension.4161819 In the calf, this increase is associated with increased tropoelastin gene expression by pulmonary artery medial SMC.'9 Similar changes are observed for several collagen types in the calf.20 Thus, the pattern of neointimal remodeling in humans at the time of surgery is different from remodeling reported in previously described animal models. Multiple factors may account for these differences between animal models of pulmonary hypertension and patients with severe pulmonary hypertension including age and species differences or duration and severity of hypertension. Previous studies on vessels from neonatal animals demonstrated that cells expressing collagen and elastin are not uniformly distributed across the vessel wall.4 This cellular complexity within the smooth muscle cell layer was not appreciated by Northern blot analysis which reflects an average of all cellular phenotypes in the vessel wall. In this respect, in situ hybridization proved invaluable for correlating cellular responses to injury in the neonatal calf model of hypoxic pulmonary hypertension. The current studies in human hypertensive vessels likewise demonstrate a complex pattern of regional heterogeneity. First, remodeling is sharply demarcated by the inner elastic lamina. Immunohistochemical stains detected cellular fibronectin and type procollagen protein only in the neointima. Likewise, in situ hybridization detected cells

expressing tropoelastin, fibronectin, type collagen and thrombospondin mRNA nearly exclusively in the neointima although some signal was observed in other regions of the vessel as well. Whether this regional heterogeneity reflects limited diffusion of exogenous growth or differentiation factors derived from infiltrating plasma, regional heterogeneity in endogenous growth factor synthesis, differences in medial and neointimal cell responsiveness to growth factors, or some other influence is currently unknown. Second, not all cells within the neointima express mRNA for a given extracellular matrix protein. Whether a single neointimal cell subpopulation is expressing all matrix genes or whether several cellular subpopulations each express a single matrix gene is again unknown. Future studies may provide insight into this previously unappreciated complexity. Neointimal tropoelastin gene expression is somewhat surprising since tropoelastin gene expression is typically limited to a brief perinatal window of intense elastogenesis during late gestation and early neonatal life.2144 Human aortic elastogenesis appears essentially complete by the end of the first decade of life.25 A clear correlation between donor age and rates of elastin synthesis also is observed in vitro in cultured elastin-producing cells with maximal elastogenesis occurring in cells from perinatal donors.24,2628 Precedent for reactivation of elastin synthesis, however, is found in both an animal model of emphysemae and in patients with emphysema.' In summary, all human pulmonary arteries obtained at the time of single-lung transplantation for severe, unexplained pulmonary hypertension were atherosclerotic. Although medial remodeling, similar to that reported in animal models, may be the initial response to developing pulmonary hypertension, remodeling at the time of surgery is characterized, to a large extent, by neointimal extracellular matrix gene expression and protein synthesis. Furthermore, these studies provide evidence that these hypertensive vessels are actively remodeling despite the chronicity of disease.

Acknowledgments The authors thank Dr. Edmond C. Crouch for helpful suggestions.

References 1. Fishman AP: Pulmonary Hypertension and Cor Pulmonale. Pulmonary Diseases and Disorders. Edited by Fishman AP. New York, McGraw-Hill, 1988, pp. 999-1048 2. Reid L: Vascular remodeling. The Pulmonary Circulation:

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Normal and Abnormal. Edited by Fishman AP. Philadelphia, U. of Pennsylvania Press, 1990, pp. 259-282 3. Heath D, Wood EH, DuShane JW, Edwards JE: The relation of age and blood pressure to atheroma in the pulmonary arteries and thoracic aorta in congenital heart disease. Lab Invest 1960, 9:259-272 4. Prosser IW, Stenmark KR, Suthar M, Crouch EC, Mecham RP, Parks WC: Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization. Am J Pathol 1989,135:1073-1087 5. Wrenn DS, Mecham RP: Immunology of Elastin. Methods of Enzymology 1987,144:246-259 6. Vartio T, Laitinen L, Narvanen 0, Cutolo M, Thornell L-E, Zardi L, Virtanen I: Differential expression of the ED sequence-containing form of cellular fibronectin in embryonic and adult human tissues. J Cell Sci 1987, 88:419-50 7. Prater CA, Platkin J, Jaye D, Frazier WA: The properdin-like type repeats of human thrombospondin contain a cell attachment site. J Cell Biol 1991, 112:1031-1040 8. Kuhn C l1l, BoldtJ, King TEJr, Crouch E, VartioT, McDonald JA: An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis 1989,140:1693-1703 9. Crouch E, Quinones F, Chang D: Synthesis of type IV procollagen in lung explants. Am Rev Respir Dis 1986, 133:618-625 10. Indik Z, Yeh H, Ornstein-Goldstein N, Sheppard P, Anderson N, Rosenbloom JC, Peltonen L, Rosenbloom J: Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA. Proc Nat Acad Sci 1987, 84:5680-84 11. Kornblihtt ARK, Umezawa K, Vibe-Pederson, Baralle FE: Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J 1985, 4:1755-1759 12. Hennessy SW, Frazier BA, Kim DD, Deckwerth TL, Mona Baumgartel D, Rotwein P, Frazier WA: Complete thrombospondin mRNA sequence includes potential regulatory sites in the 3' untranslated region. J Cell Biol 1989, 108:729-736 13. Heath D, Edwards JE: Configuration of elastic tissue of pulmonary trunk in idiopathic pulmonary hypertension. Circulation 1960, 21:59-62 14. Wissler RW, Vesselinovitch D: Atherogenesis in the pulmonary artery. The Pulmonary Circulation: Normal and Abnormal. Edited by A.P. Fishman. Philadelphia, U. Pennsylvania Press, 1990, pp. 245-255 15. Heath D, Wood E, Dushane J, Edwards J: The structure of the pulmonary trunk at different ages and in cases of pulmonary hypertension and pulmonary stenosis. J Pathol Bacteriol 1959, 77:443

16. Rabinovitch M, Gamble W, Nadas A, Miettinen OS, Reid L: Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol 1979, 236:H818-H827 17. Meyrick B, Reid L: Hypoxia-induced structural changes in the media and adventitia of the rat hilar pulonary artery and their regression. Am J Pathol 1980,100:151-178 18. Kerr JS, Riley DJ, Frank MM, Trelstad RL, Frankel HM: Reduction of chronic hypoxic pulmonary hypertension in the rat by beta-aminopropionitrile. J Appl Physiol 1984, 57:1760-1766 19. Mecham RP, Whitehouse LA, Wrenn DS, Parks WC, Griffin GL, Senior RM, Crouch EC, Stenmark KR, Voelkel NF: Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension. Science 1987, 237:423-426 20. Crouch EC, Parks WC, Rosenbaum JL, Chang D, Whitehouse L, Wu L, Stenmark KR, Orton EC, Mecham RP: Regulation of collagen production by medial smooth cells in hypoxic pulmonary hypertension. Am Rev Respir Dis 1989, 140:1045-1051 21. Dubick L, Rucker RB, Last JA, Lollini LO, Cross CE: Elastin metabolism in rodent lung. Biochim Biophys Acta 1981, 672:303-306 22. Cleary EG, Sandberg LB, Jackson DS: The changes in chemical composition during development of the bovine nuchal ligament. J Cell Biol 1983, 33:469-479 23. Barrineau LL, Rich CB, Foster JA: The biosynthesis of tropoelastin in chick and pig tissues. Connect Tissue Res 1981, 8:189-191 24. Davidson JM, Shibahara S, Smith K, Crystal R: Developmental regulation of elastin synthesis. Connect Tissue Res 1981, 8:209-212 25. Berry CL, Looker T, Germain J: Nucleic acid and scleroprotein content of the developing human aorta. J Pathol 1972, 108:265-274 26. Mecham RP, Lange G, Madaras J, Starcher B: Elastin synthesis by ligamentum nuchae fibroblasts: Effects of culture conditions and extracellular matrix on elastin production. J Cell Biol 1981, 90:332-338 27. Eichner R, Rosenbloom J: Collagen and elastin synthesis in the developing chick aorta. Arch Biochem Biophys 1979,

198:414-423 28. Rucker RB, Dubick MA: Elastin metabolism and chemistry: Potential role in lung development and structure. Environ Health Perspect 1984, 55:179-191 29. Kuhn Cl, Yu SY, Chraplyvy M, Linder HE, Senior RM: The induction of emphysema with elastase. II. Changes in connective tissue. Lab Invest 1976, 34:372-30 30. Fukuda Y, Masuda Y, Ishizaki M, Masugi Y, Ferrans VJ: Morphogenesis of abnormal elastic fibers in panacinar and centriacinar emphysema. Hum Pathol 1989, 20:652-659