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(Pediatrics in Review. 1999;20:e91-e102.)
© 1999 American Academy of Pediatrics
OBJECTIVES
After completing this article, readers should be able to:
Introduction
Pulmonary hypertension may arise in the neonate and young infant from a multitude of disease processes involving both the cardiac and respiratory systems. Common causes include congenital heart disease with increased pulmonary blood flow, diaphragmatic hernia with associated lung hypoplasia, bronchopulmonary dysplasia, and idiopathic persistent pulmonary hypertension of the newborn. Regardless of the cause, chronic pulmonary hypertension is associated with maladaptive changes in pulmonary vascular structure and function (remodeling). These changes prompt further rises in pulmonary artery pressure, limit responses to vasodilator therapies, and if persistent, lead to cor pulmonale and death.
Clearly, an understanding of the mechanisms contributing to vascular remodeling is necessary to design optimal treatment strategies. Unfortunately, no laboratory model of pulmonary hypertension duplicates all of the pathophysiologic changes seen in the various types of neonatal and pediatric pulmonary hypertension. However, hypoxia is a common pathologic stimulus in the neonatal period that modulates both pulmonary artery pressure and pulmonary blood flow and often is associated with pulmonary hypertension. Importantly, hypoxia-induced pulmonary hypertension in neonatal animal models reproduces many of the characteristics seen in neonates and infants who have severe pulmonary hypertension. These include severe hypoxemia with right-to-left intracardiac and ductal shunting of blood, altered pulmonary vasoreactivity, and dramatic vascular remodeling with increased pulmonary artery cellularity and synthesis and deposition of matrix protein. The data summarized in this review are derived largely from animal models of hypoxia-induced pulmonary hypertension.
Blood vessels in the lung undergo profound structural remodeling as chronic hypoxic pulmonary hypertension develops; changes include cellular hypertrophy, hyperplasia, and increased deposition of structural matrix proteins such as collagen and elastin in the vessel wall. The phenotype of cell populations that comprise the vessel wall (endothelial, smooth muscle, and fibroblast cells) change markedly and are responsible for the alterations in structure and function. However, the cellular and structural changes observed in pulmonary hypertension vary significantly, depending on patient age, duration and degree of hypoxic exposure, and the presence of associated abnormalities such as chronic inflammation or high pulmonary blood flow. Vascular responses to hypoxia involve complex cell-cell interactions that are mediated by the release of growth factors, cytokines, and biologic messengers and by changes in the composition of interstitial and basement membrane matrix proteins. Further, the cellular responses to local decreases in oxygen concentration are heterogeneous between and even among the cell populations that comprise the vessel wall. Dramatic differences have been observed in the proliferative, matrix-producing, and secretory phenotypes of different cells along the longitudinal axis of the vessel wall as well as among cells at a specific site. In addition, evidence demonstrates that the response of a particular gene to hypoxia in vivo is regulated differentially at the level of specific cell types and regions within the tissue. Thus, up- or downregulation of specific gene expressions by unique or specialized cells within a tissue may influence the overall response of an organ or tissue to local changes in oxygen concentration. An understanding of the changes that occur in cells in the vascular wall when exposed to decreases in oxygen concentration and the mechanisms that cause them are critical for a better understanding of the pathophysiology of many vascular disorders, including neonatal and pediatric pulmonary hypertension.
Pathophysiologic Structural Remodeling of the Chronically Hypoxic Perinatal Pulmonary Vasculature
All forms of chronic pulmonary
hypertension are characterized by
both active vasoconstriction and
structural changes in the pulmonary
vascular wall that include cellular
hyperplasia and increased production
and deposition of extracellular
matrix. For example, maintaining or
exposing the newborn to hypoxic
conditions alters pulmonary vascular
structure and reactivity sufficiently
to produce severe pulmonary
hypertension. The resulting elevation of
pressure may have both reversible
(ie, vasoconstrictive) and fixed
(ie, vasodilator-unresponsive)
components. The vasoconstrictive
component predominates in the early
stage of pulmonary hypertension
when there is relatively little change
in the structure of the vessel wall
(at least at the light microscopic level).
Over time, though, the fixed
component becomes more prominent (Fig. 1
).
The pulmonary arteries fail to
dilate with the administration of
either endothelial-dependent (ie,
acetylcholine) or -independent (nitric
oxide, sodium nitroprusside,
isoproterenol) vasodilators or oxygen. The
evolution of this relatively fixed,
vasodilator-unresponsive component of
pulmonary hypertension is related
temporally to the development of
thickened vascular media and
adventitia, with dramatic increases in the
deposition of structural matrix
proteins such as collagen and elastin in
the pulmonary artery walls (Fig. 2
).
Indeed, in our model of neonatal
hypoxic pulmonary hypertension, as
pulmonary artery pressure increases
over a 14-day exposure to hypoxic
conditions, it is the increased
deposition of matrix protein in the vessel
wall that we believe coincides most
closely with the development of the
fixed component of pulmonary
hypertension.
The pulmonary circulation of infants in the immediate perinatal period may be especially vulnerable to the presence of even regional alveolar hypoxic conditions. Haworth et al have demonstrated the profound effects that perinatal hypoxia has on the structure of the neonatal pulmonary vascular bed (see Suggested Reading). When fetal levels of hypoxia were maintained postnatally in the large white pig, the fetal pulmonary circulation did not undergo the full dramatic remodeling that normally occurs after birth. Pulmonary arterial endothelial and smooth muscle cells from the animals that had been exposed to chronic hypoxia at birth maintained the shape, overlap, and interdigitation characteristics of fetal life, which resulted in an increased pulmonary artery medial thickness compared with normoxic controls. In the hypoxic neonatal calf model, the histologic structure of the small pulmonary arteries from calves made hypoxic for 1 and 3 days was very similar to that of the newborn just hours old. In addition, the pulmonary artery resistances and pressures in the hypoxic animals failed to regress. Thus, instead of the normal thinning of the pulmonary arterial wall and increase in vascular lumen diameter that results in a concomitant fall in pulmonary artery pressure and resistance, the small pulmonary arteries of calves remained thickened and had evidence of increased deposition of pulmonary vascular extracellular matrix after 2 weeks of exposure to hypoxia. These findings are consistent with those described in human neonates who have pulmonary hypertension in whom there is a significant element of fixed pulmonary hypertension.
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Alterations in Endothelial Cells During Development of Chronic Hypoxic Pulmonary Hypertension
The endothelium forms a nonthrombogenic, semipermeable barrier between the blood stream and all extravascular tissues and fluid compartments in the body. In addition, it influences vascular tone, hemostasis, growth, differentiation, chemotaxis, and the response of other vascular compartments to injury. Given this important role in such a wide array of vascular functions, it is not surprising that significant adaptations occur in the structure and function of the endothelial cell during the development of hypoxic pulmonary hypertension.
HISTOLOGY/MORPHOLOGY
Several studies have characterized
the histologic changes that occur in
the endothelial cells of both large
and small vessels in response to
chronic hypoxic exposure. Hypoxia
increases intimal thickness by
causing hypertrophy and hyperplasia in
both the endothelial and
subendothelial layers. Endothelial cell
hypertrophy is associated with increased
numbers and size of cell organelles,
including ribosomes, rough
endoplasmic reticulum, and golgi
apparatus. Increases in DNA synthesis and
cell number are demonstrated by the
approximately three-fold increase in
3H-thymidine incorporation observed
early in the course of hypoxic
exposure. Diffuse subendothelial edema
also occurs. The number of
pinocytotic vesicles increases, as do the
intracellular gaps between
endothelial cells. There is often focal
disruption and lysis of the endothelial
cell basement membrane that creates
a patchy appearance of microfibrillar
material in the thickened
subendothelial layer much like that reported
in the aorta of hypertensive rats. In
addition, the presence of collagen
fibers, elastin, and microfibrils in
the subendothelial space, internal to
the endothelial cell basement
membrane, suggests an increase in the
production of these proteins by the
endothelial cell. Under normal
conditions, it appears that endothelial
cell production of elastin is
suppressed sometime in late fetal or
early neonatal life. It is very likely
that the endothelial cell re-expresses
tropoelastin mRNA in response to
injury. In addition, smooth muscle
cells (SMC) are recruited into this
enlarging subendothelial space,
which could contribute to the
accumulation of protein in this space.
CHANGES IN GROWTH FACTOR
AND VASOACTIVE SUBSTANCE PRODUCTION
Hypoxia disturbs endothelial
function by altering the regulation of
vascular tone, increasing
permeability, reducing the antithrombotic
activities of the endothelium, and
promoting release of cytokines and
growth factors. The relationship
between the vasoconstrictor response
to acute hypoxia and the structural
remodeling characteristic of chronic
hypoxia is not understood. The onset
of hypoxia that is associated with
acute changes in vasoreactivity and
sustained hypoxia influences the
activity of certain transcription
factors for hypoxia-sensitive genes,
several of which promote cell
growth. Table 1
lists many of the
growth- and tone-modulating
substances synthesized by the vascular
endothelium whose production is
known or believed to be influenced
by hypoxia.
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The endothelium helps to regulate the tone and growth of the underlying vascular SMC through continuous production of various vasoactive mediators. Chronic exposure to hypoxia reduces the production of both prostacyclin and nitric oxide (NO). Hypoxia also can inhibit uptake of the NO precursor L-arginine by pulmonary arterial endothelial cells within 4 hours and may suppress expression of NO synthase and activity. Although inhibition of prostacyclin and NO is not believed to cause hypoxic vasoconstriction, both substances are antiproliferative, with NO acting via cGMP and prostacyclin through a cAMP-mediated effect. Thus, a reduction in their production could contribute to proliferation of SMC or fibroblasts.
Hypoxia may modulate the inhibitory effect of endothelial cells on vascular cell proliferation by other mechanisms. NO can induce reversible inactivation of the protein kinase-C (PKC) pathway, an intracellular signaling pathway associated with cell proliferation. Normal endothelial cells also secrete heparan sulfate, which directly inhibits growth of SMC by a posttranslational PKC-dependent mechanism. Hypoxia has been shown to inhibit release of heparan sulfate from endothelial cells, thereby countering the normal endothelial mechanisms that act to restrain proliferation of SMC.
The contribution of vascular endothelial growth factor (VEGF) to the hypoxic response is not yet clear, although VEGF, VEGF mRNA, and the transcripts for its receptors are upregulated in chronic hypoxia. VEGF may be important in regulating release of NO. It also increases vessel permeability and can stimulate prostacyclin synthase. Although VEGF and its receptors have been located on the pulmonary vasculature, most of the mechanistic studies have not been carried out on pulmonary vascular endothelial cells.
Vasoconstrictor substances produced by the endothelium include endothelin, thromboxane A2, angiotensin II, prostaglandin H2, leukotrienes, platelet activating factor, and superoxide anion. Hypoxia is associated with an increase in the plasma level of endothelin (ET)-1 and increased gene transcription for ET-1 and for the ETA receptor on the SMC. It also increases the synthesis and release of angiotensin-converting enzyme (ACE) and stimulates the uptake of serotonin in pulmonary arterial endothelial cells. Several powerful vasoconstrictors, such as ET and angiotensin, also promote SMC growth, and some can enhance growth further by stimulating the production of other growth factors. Thromboxane A2 and angiotensin II, for example, stimulate and increase the expression of basic fibroblast growth factor and insulin-like growth factor-I, respectively. ACE activity is reduced in chronic hypoxic rat whole lungs, but ACE protein and gene expression is upregulated in newly muscularized peripheral pulmonary arteries. ACE inhibition with captopril attenuates muscularization of pulmonary vessels and the severity of the pulmonary hypertensive response to hypoxia. Also, stimulation of complementary signal transduction pathways by different stimuli can have pronounced synergistic effects. PKC and mitogen-activated protein kinase (MAPK) appear to play central roles in the interaction of mechanical, hypoxic, and growth factor-induced responses.
Hypoxia also increases the expression of several cytokines and growth factor genes in vitro and in vivo. The expression of platelet-derived growth factor-B is increased in the hypertensive lungs of hypoxic rats. Interleukin-1a (IL-1a)-dependent upregulation of ICAM-1 is seen in pulmonary microvessels of intact hypoxic mice, and hypoxia-induced increases in IL-8 were mimicked by increases in IP-10 (murine homologue) in hypoxic mice. The mechanism by which hypoxia upregulates responsive genes is not clear. However, the transcription factor known as hypoxia-inducible factor-1 (HIF-1) is induced in all cell types tested, and the hypoxic response elements of target genes such as VEGF, erythropoietin, and several glycolytic enzymes are recognized and regulated by this transcription factor. HIF-1 is redox-sensitive. Activation of several hypoxia-related genes appears to involve a heme protein, possibly acting as an oxygen sensor. These include the genes that encode for ET-1, VEGF, tyrosine hydroxylase, and erythropoietin.
Thus, by regulating vascular tone, permeability, and production of growth factor, the endothelium plays a central role in modulating the pulmonary vascular response to hypoxia and ultimately the pathologic structural remodeling that occurs during the development of chronic pulmonary hypertension. There is now an overriding need to understand how to direct and manipulate the endothelial cell signal transduction pathways that control the potentially protective, beneficial effect of certain vasoactive substances and the signaling pathways that control structural remodeling.
Response of Endothelium to Hypoxia-induced Hemodynamic Stress
Exposure to acute or chronic hypoxia elicits significant changes in the hemodynamic stresses imposed on the pulmonary circulation. It is difficult to separate the cellular responses induced by hypoxia from hemodynamic forces in vivo because they may act cooperatively or in some instances synergistically. Thus, to comprehend the remodeling associated with chronic hypoxia, the effects of hemodynamic forces, particularly sheer stresses, on the endothelium must be understood. Blood flow is an important acute modulator of vascular tone, but it also can influence the more chronic process of vascular remodeling. Many flow-induced effects result, at least in part, from the ability of the endothelium to sense and transduce hemodynamic stimuli into changes in vascular structure and function. Examples include increased micromolecular permeability, lipoprotein accumulation, leukocyte adhesion molecule expression, mononuclear leukocyte recruitment, and vessel size changes following alterations in SMC proliferation or matrix protein synthesis.
Almost all available evidence tends to support the hypothesis that the endothelium is the site of sheer stress transduction in the arterial wall. Multiple endothelial sheer sensors elicit responses via interlinked signal transduction pathways. Recent work suggests sheer sensing at focal adhesion plaques and at G protein-coupled receptors. However, sheer stress-sensitive ion channels also have been identified. It is not surprising that sheer stress drives multiple signal transduction cascades, given the spectrum of physiologic responses and the large panel of genes that sheer regulates, probably via a large array of transcriptional regulators.
Endothelial cells not only transduce sheer stress and elaborate a number of genes, including vasoactive and growth-promoting cytokines that induce remodeling of the arterial wall, but the response to most hemodynamic stresses begins at the level of the endothelium. It appears that cell shape, orientation, and cytoskeletal organization all are sensitive to sheer stress. Although this morphologic sensitivity is well known, its regulation is poorly understood. Changes in cell shape induced by sheer stress have potentially important implications for physiologic functions of the endothelium. They necessitate the partial disassembly and reassembly of the adherence junction, a cadherin-based protein complex that mediates cell-cell adhesion. Reassembly is not completed until 24 to 48 hours after sheer stress is imposed. Disruption of the adherence junction compromises the endothelial permeability barrier, which may help to explain the high permeability of endothelium seen at vascular sites exposed to altered flow conditions. Changes in permeability are believed by some to be a critical first step in initiating the remodeling process.
The structural changes associated with long-term alterations in blood flow involve alterations in the tissue content of the vessel wall and its reorganization. Depending on the pathophysiologic condition, there may be increases in the number of SMC and significant changes in the content of matrix protein. Reorganization of the critical matrix protein elastin and net accumulation are important in vascular remodeling. It appears that modifications in vessel diameter elicited by hemodynamic changes are due to sheer stress-induced alterations in endogenous vascular elastases as well as changes in matrix metalloproteases (MMP), including MMP-2 and MMP-9. Thus, the endothelium seems to have profound effects on the structure and composition of the vessel wall under changing hemodynamic conditions. It is not surprising, therefore, that hypoxia and changes in blood flow may act synergistically under certain conditions to cause dramatic structural alterations in the pulmonary circulation. This has important clinical relevance in that certain conditions characterized by high pulmonary blood flow may be exacerbated by local hypoxic conditions within the lung to cause dramatic structural remodeling.
Alteration in SMC Function and Phenotype
The severity of chronic hypoxic pulmonary hypertension is determined, at least in part, by the extent of structural changes in the medial compartment of the pulmonary arterial wall. These changes include proliferation of SMC, hypertrophy, and deposition of matrix protein. Hypoxia, mechanical stress, and blood-borne and locally produced mitogens act collectively to drive these cellular responses. They activate a cascade of intracellular signaling mechanisms, including tyrosine kinases, calcium (Ca++), MAPK, and PKC, that promote growth of SMC and synthesis of matrix protein. Synergy between different stimuli and resulting "crosstalk" between signal transduction pathways augments the extent of vascular changes. Susceptibility to these stimuli is enhanced when inhibiting mechanisms are impaired, such as endothelial barrier function, local production of heparan sulfates, and prostacyclin- and NO-induced increases in cyclic nucleotides. Intrinsic (developmental, genetic, acquired) differences in growth and matrix synthetic capacity and local and regional phenotypic heterogeneity of pulmonary artery SMC also regulate the pattern of remodeling in the tunica media in response to chronic hypoxia.
HISTOLOGY/MORPHOLOGY
Increases in the thickness of the
medial layer of normally muscular
arteries and an extension of muscle
into smaller and more peripheral
vessels are common to all forms of
human pulmonary hypertension.
Detailed characterization of the
changes in SMC induced by chronic
hypoxia have been examined in
animal studies.
The timing and nature of changes in SMC of both the proximal and distal pulmonary vasculature differ. In the proximal pulmonary arteries of adult rats, the media thickens due to hypertrophy and hyperplasia of the individual muscle cells and increased synthesis and deposition of extracellular matrix proteins, elastin, and collagen. In the distal vasculature, the muscularization of previously nonmuscular arteries or so-called "extension" is brought about by differentiation and hypertrophy of cells (intermediate cells and pericytes) present in the wall. In addition, some investigators believe that interstitial fibroblasts are recruited locally into a cell that exhibits muscle-specific proteins and functions. Both the intermediate cell and the pericyte undergo significant changes and acquire a more smooth muscle-like appearance. These cells produce a network of elastin, which appears to induce the formation of a new elastic lamina between the muscle layer and the endothelium. Because this internal lamina is not as complete as in normal muscular arteries, the endothelial cell and new muscle cells form frequent contacts. These contacts differ from those observed in normal muscular pulmonary arteries, suggesting that a close and perhaps different communication might exist between these cells in the newly muscularized vessels.
If hypoxic exposure occurs at or around the time of birth, different cellular responses are noted than if hypoxia occurs later in life. The peripheral pulmonary arteries of newborns who die in the first 36 hours after a hypoxic event demonstrate an extremely thick-walled fetal-like structure. The SMC of all animals exposed to hypoxia during the first week of life demonstrate an increased concentration of myofilaments. In the large arteries, the adluminal SMC (on the outside of the vessel) exhibit a greater increase in myofilaments than do the abluminal cells (on the inside of the cell). This very rapid and dramatic response in neonates requires a much longer period of exposure to elicit in older animals.
EFFECT ON VASCULAR MATRIX PROTEIN PRODUCTION
Marked increases in deposition of
matrix protein are noted in the
hypoxic neonatal pulmonary
vascular wall. The deposition of matrix
proteins in blood vessel walls is
crucial for normal blood vessel
structure and function, and an increase
could influence dramatically the
vascular response to changes in
hemodynamics and other stimuli. Because
of its importance, the complex
pattern of structural matrix protein
expression is tightly regulated
throughout development. For
example, tropoelastin mRNA is highly
expressed in the blood vessel wall
primarily during late fetal and early
neonatal life, but it decreases rapidly
with increased maturity until
virtually no expression is detected in
adult vessels. Further, regulation of
matrix protein expression during
development may differ even within
a tissue such as the pulmonary
vasculature. Such differences may vary
along the longitudinal axis of the
pulmonary artery and, as will be
discussed, vary between specific
subpopulations of SMC within the
same pulmonary artery wall.
We investigated the hypothesis that maintaining high pulmonary artery pressure, as occurs in the hypoxic neonatal calf model of pulmonary hypertension, might change the normal postnatal pattern of extracellular matrix protein expression in newborn pulmonary arteries. Such changes ultimately would result in excessive production of new protein that would contribute to pathologic pulmonary arterial remodeling and subsequent pulmonary hypertension that is unresponsive to conventional therapies. To investigate this hypothesis, we examined the normal developmental expression of three extracellular matrix proteins—fibronectin, tropoelastin, and alpha-1 (I) procollagen and fibronectin—in small resistance-sized pulmonary arteries of both fetal and neonatal calves and assessed the impact of severe pulmonary hypertension induced by hypoxia on their expression. In situ hybridization was used to localize and assess expression of these matrix mRNAs.
The developmental regulation of
tropoelastin mRNA expression is
both tissue- and species-specific. For
example, tropoelastin mRNA
expression and elastin deposition peak
postnatally in the rat, which is born
with relatively immature lungs. In
contrast, tropoelastin mRNA
expression in small resistance pulmonary
arteries occurs late in gestation in
the calf, which is born with more
mature lungs. In the neonatal calf
model of hypoxic pulmonary
hypertension, small muscular pulmonary
arteries, which had ceased
tropoelastin expression in normal, normoxic
animals at birth, re-expressed
tropoelastin mRNA postnatally in
response to hypoxia (Table 2
).
The strongest tropoelastin mRNA signal
in small resistance pulmonary
arteries localized to the outer medial and
adventitial layers of pulmonary
hypertensive arteries. Thus,
maintaining hypoxia early in the
postnatal period resulted in the abnormal
re-expression of tropoelastin mRNA
in pulmonary arteries that had
ceased expression in utero. This
expression ultimately results in
increased elastin deposition in the
vessel wall as it undergoes
pathologic vascular remodeling.
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Type I collagen is a rigid
structural matrix protein that does not
appear to be necessary for normal
early lung development. We
detected little or no alpha-1 (I)
procollagen mRNA in muscular
pulmonary arteries from late-gestation fetal
calves or during normal postnatal
life, although expression was present
in large elastic arteries and in the
lung interstitium (Table 2
).
However, during exposure to chronic
hypoxia, alpha-1 (I) procollagen
expression was induced rapidly in
the muscular pulmonary artery.
Because of the high tensile strength
of collagen, the increase in alpha-1
(I) procollagen expression and
subsequent protein deposition increase
the ability of the pulmonary artery
to withstand the higher vascular
pressure and flow that accompany
the development of hypoxic
pulmonary hypertension. This same
process, however, may alter the
reactivity of the vessel and restrict its
ability to vasodilate in the face of
higher pressure and flow, as occurs
when neonatal calves and other
experimental animals are exposed to
chronic hypoxia.
Fibronectin is a 460-kd
glycoprotein dimer that helps to control cell
migration, division, and
differentiation. In accordance with its
important role during embryologic
development, we found very high
fibronectin mRNA levels in
late-gestation fetal calf pulmonary
arteries (Table 2
). Postnatally, these
levels decreased and became
undetectable by 15 days of life. The
late fetal pattern of fibronectin
expression persisted in small
pulmonary arteries from chronically
hypoxic animals, with the mRNA
signal localized predominantly to the
outer medial and adventitial areas of
the pulmonary artery wall. Because
fibronectin maintains cells in a more
synthetic and proliferative
phenotype, the persistence of such
expression in the pulmonary arteries of
chronically hypoxic neonatal
animals that have pulmonary
hypertension might maintain or induce cells
in the outer media to remain highly
proliferative, mobile, and capable of
rapidly increasing matrix protein.
These cells, therefore, would be
more likely to respond rapidly to
changes in pulmonary vascular
pressure and blood flow with increased
proliferation and structural protein
synthesis and deposition.
The development of pulmonary hypertension also may alter the expression of proteolytic enzymes and other matrix proteins that have growth-regulating activities. The proteolytic activity of elastases and metalloproteinases in the vessel wall has been shown in both cell culture and whole animal studies to induce the release of mitogenic growth factors from the extracellular matrix. In addition, upregulation of the matrix glycoprotein tenascin, which has been identified in both humans and experimental animals that have pulmonary hypertension, is accompanied by the proliferation of SMC. Interestingly, the tenascin effect on cell proliferation appears to be regulated by elastase and metalloproteinases; that is, when collagen is proteolyzed, integrin binding sites become exposed. When bound, tenascin gene transcription is induced via MAPK. It is of interest that when MMP or elastases are inhibited, hypertrophied pulmonary arteries regress in organ culture. This is associated with downregulation of expression of tenascin.
UNIQUE RESPONSES OF SPECIFIC
SMC SUBPOPULATIONS TO HYPOXIA
The vascular media (at least of large
vessels) is not composed of a single
population of SMC, but rather is a
mosaic of phenotypically distinct
subpopulations. New investigations
are examining the hypothesis that
specific subsets of arterial SMC
contribute selectively to the vascular
response to injury. A substantial
body of experimental evidence
demonstrates that pathologic lesions in
atherosclerosis, restenosis, and
hypertension in humans (as well as
the neointima in injured animal
vessels) are composed primarily of cells
that have nonmuscle-like
characteristics. The absence or paucity of
muscle-specific markers in these
cells historically had been attributed
to a process of "phenotypic
modulation" of a differentiated medial SMC
type during its proliferation and
migration into the intimal space.
However, the identification of cells
with nonmuscle-like characteristics
in the normal mature vascular media
indicate that pathologic lesions in
the arteries also could originate via
expansion of a subset of relatively
"undifferentiated" or "immature"
medial cells. For example, it has
been suggested that the intimal
thickening seen after balloon injury
in canine arteries results from
selective proliferation of a subset of
nonmuscle-like cells in the arterial
media. Recent studies demonstrating
the monoclonality of atherosclerotic
plaques in human arteries also raised
the possibility that unique subsets of
medial cells participate in the
pathologic response of the vessel wall to
injury.
We found marked heterogeneity in the proliferative and matrix-producing responses of pulmonary medial SMC to the stimuli associated with hypoxic pulmonary hypertension. Using double-label immunofluorescence staining, phenotypically unique subpopulations of SMC in bovine pulmonary arteries demonstrated markedly different proliferative responses to hypoxia-induced pulmonary hypertension. At every posthypoxic time point studied, greater than 95% of the proliferation occurred within a subpopulation that had no expression of the muscle-specific marker metavinculin; whereas a population of SMC expressing metavinculin remained quiescent. These findings argue that the population of metavinculin-negative cells is functionally distinct from a metavinculin-positive population of cells. This argument is strengthened when the matrix protein expression activity of these subpopulations of SMC is assessed. In the normal bovine pulmonary artery, a subpopulation of SMC acquires the ability to express metavinculin during development. These metavinculin-expressing cells no longer express tropoelastin (TE), while nonmetavinculin-expressing cells continue to express TE mRNA. Indeed, even with the development of hypoxic pulmonary hypertension during which TE expression is greatly increased in other subpopulations of SMC, the subpopulation of metavinculin-positive cells does not express TE.
Initial observations in the systemic circulation of two populations of SMC that had markedly different morphologies and growth patterns (ie, one isolated from the neointima of injured rat carotid arteries and the other from the unmanipulated tunica media) supported the hypothesis that the arterial wall may be composed of different types of SMC. It also is possible that neointima originates via expansion of a small pre-existing medial population of SMC that has enhanced potential for proliferation and extracellular matrix protein synthesis rather than simply by phenotypic modulation of the overall medial population of SMC. Investigators recently demonstrated that two morphologically and functionally distinct types of SMC could be isolated from different compartments of the vascular media at the same site in the normal rat aorta. Aortic medial cells (MSMC) were isolated from the media after removal of the intima and adventitia. Aortic intimal cells (ISMC) were isolated from the luminal side of everted rat aortas by scraping. MSMC were spindle-shaped, grew in the hill-and-valley pattern traditionally described for differentiated vascular SMC, and expressed alpha-SM-actin and SM-myosin. Conversely, ISMC displayed a polygonal or epitheloid shape, grew mainly as a monolayer, expressed alpha-SM-actin but not SM-myosin, and were negative for Factor VIII antigen. ISMC produced large amounts of a laminin- and type IV collagen-rich extracellular matrix, which had a unique and characteristic pericellular distribution. Contractile responses to ET differed significantly between the two populations. Interestingly, the ISMC isolated from the normal rat arteries exhibited characteristics very similar to those reported for neointimal cells isolated from injured carotid arteries.
We also have performed cell culture studies using pulmonary and systemic arteries from the neonate of a large mammalian species (bovine), with the goal of isolating and maintaining in culture heterogeneous arterial subpopulations of SMC that exhibited unique characteristics similar to those observed in vivo. We isolated four phenotypically unique populations, each exhibiting distinct morphological and biochemical characteristics. The isolated cell populations could be split broadly into two major categories that exhibited either smooth muscle characteristics or "nonmuscle-like" features. Interestingly, the cell subpopulations isolated from the bovine species demonstrated many morphological, biochemical, and functional characteristics similar to cell subpopulations derived from the normal rat arterial media. As was the case with the rat cells, the observed morphological and biochemical differences were maintained by the distinct cell populations over multiple passages in culture. None of the populations modulated into another phenotype, supporting the possibility that they represented distinct cell lineages.
Cells exhibiting unique morphological and biochemical characteristics also differed significantly with respect to their proliferative responses to growth-promoting stimuli. In general, the "nonmuscle-like" cell subpopulations exhibited markedly enhanced growth capabilities under serum-stimulated conditions compared with "differentiated" populations of SMC. Moreover, nonmuscle-like cells exhibited the capacity to grow in plasma-based media; the differentiated SMC remained quiescent. We also found specific subsets of cells among the nonmuscle-like cell populations that proliferated autonomously, similar to embryonic aortic cells.
Because in vivo studies have demonstrated selective proliferative responses of arterial SMC to hypoxia-induced pulmonary hypertension, we examined the possibility that distinct cell subpopulations isolated from the normal arterial media also would exhibit different responses to hypoxia in vitro. Indeed, only specific cell populations proliferated in response to hypoxia (in general, the nonmuscle-like cells) compared with differentiated SMC, whose growth was inhibited under hypoxic conditions.
SIGNALING PATHWAYS IN SMC SUBPOPULATIONS
The remarkable differences in
proliferative and matrix protein synthetic
responses to similar stimuli between
different medial subpopulations of
SMC suggest that the membrane
receptor or intracellular signaling
pathways controlling these responses
may differ substantially. The
contribution of the receptor tyrosine
kinase and the G protein-coupled
pathways to growth vary between
vascular SMC isolated from
different species. For example, cells
isolated from the subendothelial space,
which exhibit markedly enhanced
growth potential, use a pertussis
toxin-sensitive G protein-coupled
pathway that does not appear to
contribute to growth in the other cell
types in the bovine arterial wall.
Additional studies using stable
transfection have shown that
overexpression of specific G proteins in
vascular cells significantly changes the
functional responses of the cell to
external stimuli.
Even in the absence of obvious receptor or membrane differences, cells could exhibit different responses to similar stimuli based on the presence of or differential activation of downstream effector pathways. Recently it was demonstrated that the MAPK pathway can mediate either growth inhibition or proliferation in different human vascular types of SMC, depending on the availability of specific downstream targets. Some human vascular SMC expressed the inducible form of cyclo-oxygenase (COX-2), and other SMC did not. In those cells that expressed COX-2, activation of MAPK served as a negative regulator of proliferation, in contrast to SMC that lacked COX-2, in which MAPK activation led to proliferation. In cells expressing COX-2, platelet-derived growth factor-induced MAPK activation led to cytosolic phospholipase A2 activation, prostaglandin E2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which strongly inhibits SMC proliferation. Thus, the biologic outcome in response to similar stimuli, at least in SMC mediated by MAPK, is highly dependent on downstream enzymes expressed by the cell, which is not unexpected. It is interesting that the only subpopulations of SMC that appear to exhibit enhanced proliferative responses to hypoxia, in the absence of exogenous mitogens or serum, are those that demonstrate constitutive activation of extracellular signal-regulated kinase (Erk) 1 and 2.
Another intracellular signaling pathway that exerts a wide variety of effects on different cell processes is the PKC pathway. Numerous isozymes of PKC are regulated both developmentally and in a cell-specific manner. Thus, specific PKC isozymes and their susceptibility to activation could confer unique properties to different cell types. Because PKC activation can contribute to a general pattern of overall enhanced growth capacity, we tested whether the cell subpopulations susceptible to hypoxic growth stimulation would exhibit different patterns of PKC isozyme expression than those whose growth was inhibited by hypoxia. Further, because the alpha-isozyme of PKC (a calcium-dependent isoform) is an important determinant of hypoxic growth capacity, we compared the level of expression of PKC-alpha isoform in the two medial SMC subtypes and found that nonmuscle-like cells had increased levels of immunodetectable PKC-alpha compared with the nonproliferative differentiated SMC. This pattern of isozyme expression was paralleled by increased whole cellular PKC catalytic activity in the hypoxia-sensitive compared with hypoxia-insensitive cells. Thus, distinct arterial cell subpopulations, similar to those observed in vivo, were isolated and maintained in culture and demonstrated unique differences in the signaling mechanisms that appear to contribute to their unique growth responses.
Alterations in Fibroblast Function and Phenotype
HISTOLOGY/MORPHOLOGY
In animal models, the earliest and
most dramatic structural changes
following hypoxic exposure are
found in the adventitial compartment
of the vessel wall (Fig. 2
). Resident
adventitial fibroblasts exhibit early
and sustained increases in
proliferation that exceed those observed in
endothelial cells or SMC. In
addition, there are early and dramatic
changes in extracellular matrix
protein synthesis, including early
upregulation of collagen, fibronectin,
and tropoelastin mRNAs, followed
by a subsequent increased deposition
of each of these proteins (Table 2
).
These changes in the proliferative
and matrix-producing phenotype of
the fibroblast are accompanied by
the appearance of alpha-SMC actin
in some of the cells in the
adventitial compartment, indicating a
modulation of fibroblasts to
myofibroblasts. The fibroproliferative changes
in the adventitia ultimately are
associated with luminal narrowing and a
progressive decrease in the ability of
the vessel wall to respond to
vasodilating stimuli. In humans, significant
adventitial fibroproliferative changes
also are noted in primary and
secondary forms of pulmonary
hypertension. Interestingly, perhaps the
most dramatic adventitial changes
are observed in infants who have
severe pulmonary hypertension.
UNIQUE RESPONSES OF ADVENTITIAL FIBROBLASTS
The possibility that hypoxia acts
directly or in unique ways on the
adventitial fibroblast in the setting
of chronic hypoxic pulmonary
hypertension is raised by previous
observations that reduced oxygen
tension (anoxia/hypoxia) results in
profound changes in fibroblast
physiology and metabolism. Cellular
anoxia is a biochemical state that is
distinct from hypoxia yet still
represents a normal physiologic condition
during wound healing. Because a
response that differs substantially
from that seen with hypoxia is
induced in anoxic fibroblasts, these
cells must possess a unique ability
to activate a different, possibly
overlapping set of genes to cope with
these different environmental
conditions. The activity of several
different transcription factors is
influenced by low oxygen tensions. In
cells that are stressed by oxygen
deprivation, NF-K B activity
increases as a result of
phosphorylation and subsequent degradation of
I K B-alpha. In other cells, low
oxygen tensions induce the transcription
of multiple members of the
basic/leucine zipper domain superfamily,
result in nuclear accumulation of
p53, or induce the activity of HIF-1.
It has been demonstrated that one
DNA binding activity induced in
hypoxic, anoxic, and cobalt-treated
fibroblasts recognizes secondary
anoxia-responsive elements and has
electrophoretic mobility similar to
that of HIF-1. Identification of a
mammalian anoxic response element
may prove useful in gene therapy
regimens for targeting expression to
physiologic situations of functional
anaerobiosis, such as during wound
healing. Interestingly, deregulation
of genes normally expressed during
anoxia is common in cancer cells
regardless of their state of
oxygenation. Thus, understanding the
molecular basis of the mammalian
anoxic regulatory pathway in
fibroblasts and how similar genes
become constitutively activated in
malignancy may lead to unique
approaches to therapeutic
intervention for vascular disease complicated
by hypoxia.
Recent observations in systemic models of vascular injury suggest that early activation and subsequent phenotypic modulation of the fibroblast is an important and perhaps ubiquitous response in the vascular remodeling that follows stress or injury. For example, a sequence of events occurring in adventitial fibroblasts of the coronary vasculature following balloon catheter-induced injury is similar to that seen in the skin wound healing process. Cell proliferation is an early phenomenon that may involve the entire adventitia of the blood vessel. The proliferation observed in the adventitia occurs earlier and is of greater magnitude than is seen in the coronary media. Subsequently, fibroblasts in the coronary artery adventitia differentiate into myofibroblast-like cells, with alpha-SM actin appearing in adventital cells as early as 3 days and reaching a maximum at 14 days. These changes in proliferation and contractile protein expression in adventitial cells are accompanied by the induction of procollagen alpha-1 mRNA and subsequent protein accumulation in the adventitial compartment. Additionally, recent studies suggest that these activated fibroblasts (? myofibroblasts) may migrate through the vessel wall and be at least partially responsible for the intimal thickening that ultimately characterizes the coronary vasculature following balloon injury.
Adventitial fibroblasts also are
essential in the remodeling changes
of venous grafts after they are
placed in the arterial system. The
appearance of myofibroblasts and
ultimately a collagenous scar may
contribute to the failure of
aortocoronary saphenous vein grafts to
undergo compensatory dilatation
when atherosclerotic lesions begin to
compromise the lumen.
Additionally, in hyperoxic models of lung
injury, fibroblasts are activated,
migrate, and acquire smooth
muscle-like characteristics in the small
pulmonary arteries. Fibroblasts in the
pulmonary artery adventitial layer,
much like SMC in the outer media,
also demonstrate dramatic increases
in tropoelastin, collagen, and
fibronectin expression during the
development of chronic hypoxic
pulmonary hypertension (Table 2
).
Fibroblasts in these settings are
suspected of being the "source" of cells
in newly muscularized vessels.
Thus, observations in both the
pulmonary and systemic circulations
suggest ubiquitous involvement of
adventitial cells in the vascular
repair process.
FIBROBLAST INTERACTIONS
WITH OTHER CELL TYPES
A large body of experimental
evidence demonstrates that fibroblasts
may exert significant phenotypic
effects on other cell types, raising
the possibility that they contribute to
the vascular remodeling process in
dynamic ways that are in addition to
direct changes in their phenotype.
The existence of dynamic and
reciprocal relationships between
fibroblasts and other cell types is well
documented, especially in the
developmental biology literature.
The biochemical identity of signal molecules that mediate mesenchymal-epithelial interactions has been investigated intensively, and it has been established that matrix proteins such as collagen, fibronectin, and proteoglycans play a prominent role. There is continuous feedback of information between cell and matrix. Specific matrix molecules interact with their receptors at the cell surface in a diverse array of cell behaviors. Fibroblasts also produce a number of soluble factors that function as paracrine regulators of proliferation, migration, and biosynthetic activity among neighboring cells (endothelial cells, SMC, epithelial cells). The biologic activity of the soluble factors and the nature of the extracellular matrix in contact with the cells are mutually interdependent, with soluble factors (eg, transforming growth factor beta, epithelial growth factor, insulin-like growth factor) exerting effects on matrix biosynthesis and the response of cells to these factors being modulated by the nature of the matrix. Thus, fibroblasts may have significant effects on neighboring vascular wall cells and could contribute in unique ways to the vascular remodeling process.
Recent experiments have demonstrated the importance of fibroblast communication in vascular injury models. For example, application of the inflammatory cytokine IL 1-beta to the adventitia induces coronary vasospasm and neointimal formation even without endoluminal manipulations. These findings are relevant to clinical settings because the accumulation of mast cells and an inflammatory reaction occur in patients who have coronary vasospasm and fatal unstable coronary syndromes, respectively. Similarly, molecules that inhibit cellular proliferation when applied to the adventitia decrease the development of intimal thickening in response to luminal injury. Thus, a substantial body of in vivo and in vitro evidence suggests a dynamic reciprocity in the interaction between adventitial fibroblasts and other vascular walls similar to the dynamic interactions between mesenchymal cells and epithelium during development.
MECHANISMS OF
HYPOXIA-STIMULATED FIBROBLAST
PROLIFERATION
As suggested previously, PKC is
one important intracellular signalling
pathway stimulated by hypoxia.
PKC has many important
downstream effectors. One important
target that may be crucial for
proliferation is the MAPK family of
enzymes. The MAPKs p44 (Erk
1) and p42 (Erk 2) are vital to
proliferation in response to growth
factors in a variety of cell types. The
growth factors, via their cell surface
receptors, initiate a series of events
that culminate in the
phosphorylation of inactive ras-GDP to active
ras-GTP. In turn, ras activates raf,
which activates MEK (MAPKK/Erk
kinase). MEK very specifically
activates Erk 1 and Erk 2 by tyrosine
and threonine phosphorylation.
Active Erks are proline-directed,
serine-threonine kinases that can
phosphorylate cytoplasmic proteins
and translocate to the nucleus where
they activate transcription factors
such as Elk-1 and genes involved in
proliferation such as c-fos.
Recent work has shown that Erk-mediated signaling is important in stress-induced proliferation via H2O2 in pulmonary arterial SMC and airway SMC. This response appears to be PKC-dependent. We sought to determine whether Erk 1 and 2 mediate proliferation in pulmonary arterial adventitial fibroblasts induced by hypoxic stress. To separate the proliferative effects of hypoxia from those of growth factors and cytokines, we performed experiments in growth-arrested, serum-deprived cells. Under these conditions, hypoxia induced an increase in DNA synthesis above normoxic levels, as measured by 3H-thymidine incorporation, in pulmonary artery adventitial fibroblasts as early as 24 hours after exposure. Continued exposure to hypoxia for 3 days resulted in increased cell density compared with cultures that were maintained in normoxic atmosphere. Thus, the proliferative stimulus of hypoxia is both early and sustained. Interestingly, we found that only 25% of cultured systemic adventitial fibroblasts that were isolated from the aortas of the same animals demonstrated hypoxia-induced increases in DNA synthesis when assayed for hypoxic induction of DNA synthesis.
We also sought to determine whether the proliferative effect of hypoxia on adventitial fibroblasts was dependent on oxygen concentration by comparing levels of DNA synthesis under oxygen concentrations ranging from 1% to 20%. We found that 3% oxygen stimulated DNA synthesis maximally, and 1% oxygen did not increase DNA synthesis. Hypoxic-induced proliferation was associated with an increase in Erk 1/Erk 2 activity, as measured by the ability of immuno-complexes to incorporate 32P label onto epithelial growth factor receptor peptides, a known substrate for Erk activity. Hypoxia induced a transient increase in Erk activity, peaking at 10 minutes and returning to basal levels at 30 to 45 minutes. The peak represented a 2.5-fold increase in activity that was 25% of the activity detected under maximal stimulation by serum. Transient increases in Erk activity have been reported with growth factor-induced proliferation of other cell types, usually peaking at 5 minutes and returning to basal at 15 minutes. Importantly, an increase in Erk activity was noted at 24 and 72 hours in cells that remained exposed to hypoxic conditions. Interruption of the Erk signaling pathway by inhibition of ras activation or MEK activation abrogated the ability of hypoxia to stimulate DNA synthesis and abolished the increase in cell density noted with sustained hypoxia under serum-deprived conditions. Thus, it appears that the ability of hypoxia to stimulate proliferation in adventitial fibroblasts under serum-deprived conditions is at least partially dependent on the Erk signaling pathway. At the moment, these findings demonstrate the role for Ca++-dependent isozymes of PKC (32 alpha and beta-II) in the augmented growth of immature fibroblasts. Therefore, we believe hypoxic-stimulated Erk activity to be at least partially dependent on PKC, a finding similar to that reported from hypoxia-induced proliferation in cardiomyocytes.
ROLE OF APOPTOSIS IN
ADVENTITIAL REMODELING
Cell number is determined within
each cell line by a balance between
proliferation and death, highly
regulated processes with numerous
checks and balances. Apoptosis
(programmed cell death) is a
physiologic form of cell death. Marked
increases in the rate of apoptosis
have been observed in SMC in vivo
during the development of intimal
thickening due to balloon injury. In
addition, SMC derived from human
coronary atheromatous plaques
exhibit a markedly elevated rate of
apoptosis in vitro. Interestingly,
hypoxia acts as a physiologically
selective agent against
apoptosis-competent cells in tumors, thus
promoting the clonal expansion of
the cells that acquire mutations in
their apoptotic programs during
tumor development. Activation of
JNK and p38 and concurrent
inhibition of Erk appears to be critical for
induction of apoptosis in some cells.
Preliminary studies have shown that different fibroblast subpopulations exhibit significant differences in their susceptibility to apoptosis on serum withdrawal as well as in response to hypoxia and cytokines. Susceptibility to apoptosis on serum withdrawal and in response to interferon-gamma and IL-1 alpha varies considerably among the cell subpopulations identified and in some instances appears correlated to serum-stimulated growth rate. Fast-growing subpopulations have significantly higher rates of apoptosis in response to serum withdrawal and cytokines than the slow-growing populations. It is not known whether hypoxia-sensitive subsets of fibroblasts have higher susceptibility to apoptosis or whether hypoxia can induce proliferation and apoptosis simultaneously in selective fibroblast subpopulations. The exact signaling mechanisms responsible for the induction of apoptosis in response to serum withdrawal, cytokines, and hypoxia in adventitial fibroblasts is not known.
Clinical Correlation and Summary
The molecular and cellular changes in the pulmonary circulation during the development of hypoxic pulmonary hypertension are complex. Pulmonary artery endothelial cells, SMC, and fibroblasts all undergo alterations of both intra- and intercellular signaling pathways, proliferation, and matrix protein synthesis in response both to hypoxia and to the changes in hemodynamic forces that global hypoxia imparts on the pulmonary arterial bed. The consequence of these complex changes is pathologic structural remodeling of the pulmonary vascular bed, which results in severe pulmonary hypertension that often is poorly or completely unresponsive to therapies and ultimately leads to cor pulmonale and death. However, as we begin to understand these complex processes better, we can develop new strategies to treat pulmonary hypertension, to avoid complications of present therapies, and ultimately to develop new therapies aimed at preventing the early cellular and molecular changes prompted by pulmonary hypertensive stimuli, such as hypoxia.
In light of our current understanding about the pulmonary hypertensive process, care must be taken to evaluate treatment strategies traditionally used to manage patients who may be exposed to hypoxia/hypoxemia in the presence of increased pulmonary blood flow. An example would be in the medical management of infants born with hypoplastic left heart syndrome. To "balance" the right ventricular output between the pulmonary and systemic circulations, many institutions now treat neonates with subatmospheric concentrations of oxygen. The resultant hypoxia increases pulmonary vascular tone, diminishes pulmonary blood flow, and allows for better perfusion of the systemic circulation. The success of this maneuver in acutely improving systemic blood pressure and perfusion and in treating acidosis is undisputed. However, over time the interaction between increased pulmonary vascular flow in the presence of increased pulmonary vascular tone due to hypoxic pulmonary vasoconstriction may result in an acceleration of pulmonary vascular structural changes. Such changes would mimic what has been described in the models of chronic hypoxic superimposed on increased pulmonary blood flow. If significant pulmonary structural changes ultimately result in pulmonary hypertension that is unresponsive to vasodilator therapies, definitive treatment of hypoplastic left heart syndrome with surgery or heart transplantation may no longer be feasible.
Unfortunately, current treatment options for severe pulmonary hypertension associated with significant vascular remodeling remains inadequate. Obviously, one problem is the frequently delayed diagnosis that leads to worsening vascular disease. Recognition of infants and children at risk of developing pulmonary hypertension is important, and evaluation of the pulmonary artery pressure is essential. Further, effective approaches to treatment must block progression of the vascular remodeling process and promote regression of established vascular changes. Circumventing the problem by the formation of new blood vessels may be another viable approach.
Fortunately, there are many potential targets for pharmacologic intervention, including extracellular matrix components, locally produced vasoregulatory and mitogenic proteins, intracellular signal transduction cascades, and cell cycle intermediates. Experimental data strongly suggest that manipulating the endogenous vascular elastases and MMP cascades initiated during the vascular remodeling process could be extremely useful at reducing and potentially even reversing the remodeling process. ET inhibitors have been particularly useful in animal models at preventing hypoxia-induced structural remodeling, but little evidence suggests that they are useful in reversing the vascular remodeling process. There also is reason to believe that ACE inhibitors or antagonists of the type I angiotensin receptor could be valuable. In addition, because entry into and progression of vascular cells through the cell cycle is considered a key event in the vascular proliferative diseases, targeting the machinery that regulates the cell cycle is one method of interrupting the "final common pathway" of many growth-promoting signals. This approach provides an attractive therapeutic tool for the prevention and perhaps even reversal of vascular proliferative diseases. These therapies have been successful at inhibiting intimal changes induced by injury in the systemic circulation, but no work has been done to date in the pulmonary circulation. It is clear that vascular remodeling must be considered when designing a therapeutic strategy for the patient who has severe pulmonary hypertension. The most promising strategies and the most favorable combinations of drugs remain to be identified.
Acknowledgments
Supported by National Institutes of Health grant #HL57144-03.
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