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(Chest. 2005;128:565S-571S.)
© 2005 American College of Chest Physicians

Pulmonary Hypertension*

Cellular and Molecular Mechanisms

Carlyne D. Cool, MD; Steve D. Groshong, MD, PhD; John Oakey, PhD and Norbert F. Voelkel, MD

* From the Department of Pathology, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, Denver, CO; Chemical Engineering Department, Colorado School of Mines, Golden, CO; and Pulmonary Hypertension Center and Pulmonary and Critical Care Medicine Division, University of Colorado Health Sciences Center, Denver, CO.

Correspondence to: Carlyne D. Cool, MD, Department of Pathology, B216, 4200 E Ninth Ave, University of Colorado Health Sciences Center, Denver, CO 80262; e-mail: carlyne.cool{at}uchsc.edu


   Introduction
TOP
 Introduction
Severe Angioproliferative...
 Severe Pulmonary Hypertension...
 References
 
The pathology of pulmonary hypertension can be conveniently divided into endothelial, smooth-muscle, and/or adventitial abnormalities, although not all compartments of the pulmonary artery wall are involved in each case of severe pulmonary hypertension. The classic lesion of severe pulmonary hypertension is the plexiform lesion, an abnormal proliferation of predominantly endothelial cells. Smooth-muscle thickening can be seen in all forms of the disease but is not a constant feature in the idiopathic (primary) form of the disease. The adventitia is often markedly remodeled in patients with certain forms of collagen vascular diseases associated with severe pulmonary hypertension, most notably scleroderma (Fig 1 ).



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  		Figure 1. A comparison of the histopathologic subtypes of pulmonary hypertension. Top left, A: Plexiform lesion from a patient with severe pulmonary hypertension demonstrating the exuberant proliferation of cells that compromise the lumen of the small pulmonary artery. Multiple slit-like vascular spaces are all that remain of the original lumen (hematoxylin-eosin, original x 400). Top right, B: Marked smooth-muscle hypertrophy/hyperplasia in a patient with mild pulmonary hypertension and emphysema (hematoxylin-eosin, original x 400). Bottom left, C: Adventitial fibrosis in a patient with scleroderma and moderate pulmonary hypertension (hematoxylin-eosin, original x 400). Bottom right, D: For comparison, a normal small pulmonary artery (hematoxylin-eosin, original x 400).

 
An exuberance of endothelial cell growth in the plexiform lesions of pulmonary hypertension was first described nearly a decade ago by Tuder et al.1 Much of this work changed our thinking about the pathogenesis of severe pulmonary hypertension, from one of vasoconstriction and subsequent hypoxic vessel remodeling to that of an angioproliferative event. The second significant observation made by Lee et al2 was the discovery of monoclonality in plexiform lesions of primary, but not secondary, pulmonary hypertension. Alternative avenues of research have begun with the concept that severe, angioproliferative pulmonary hypertension is a neoplastic disease, not simply a reparative process.

What other evidence do we have that severe pulmonary hypertension in adults is a neoplastic, angioproliferative disease? Yeager et al3 described somatic endothelial cell mutations involving transforming growth factor-ß receptor II and Bax genes within the microdissected endothelial cells of plexiform lesions from patients with severe pulmonary hypertension. Our group has described the loss of a tumor suppressor gene, peroxisome proliferator-activated receptor (PPAR)-{gamma}, in the plexiform lesions of patients with severe pulmonary hypertension.4 PPAR-{gamma} is antiproliferative, proapoptotic, and anti-inflammatory;56789101112131415 it has also been shown to inhibit angiogenesis.16 Thus, its loss in the plexiform lesion is consonant with an abnormal, uncontrolled growth of endothelial cells. Other markers of a severe angioproliferative cell phenotype in the plexiform lesion include expression of the anti-apoptotic protein, survivin (Fig 2 , left, A) and the presence of markers of proliferation (Fig 2, right, B). Previous work has demonstrated increased expression of vascular endothelial growth factor (VEGF),117 VEGF receptor 2 (kinase insert domain-containing receptor),17 angiopoietin-1,18 5-lipoxygenase,19 5-lipoxygenase activating factor,19 endothelin-1,20 HOX genes,21 and RANTES (regulated upon expression, normal T-cell expressed and secreted)22 in lungs from patients with severe pulmonary hypertension. Also described is the decreased expression of endothelial nitric oxide synthase,23 p27/kip1,24 and prostacyclin synthase.25 This is likely just a partial list of the descriptive phenotypic markers of severe pulmonary hypertension, but all lead to the conclusion that the cells that comprise the plexiform lesions are abnormal, proliferative and dysfunctional. The loss of important cell growth control mechanisms along with the abnormal expression of growth factors might allow for the clonal expansion of endothelial cells from a single cell that has acquired a selective growth advantage.



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  		Figure 2. Left, A: Immunohistochemical stain demonstrating the presence of the anti-apoptotic protein, survivin, in the plexiform lesion of a patient with severe pulmonary hypertension. Brown indicates positive staining (original x 400). Right, B. Immunohistochemical stain for MIB-1, an antibody that recognizes the cell cycle associated antigen Ki-67. Rare cells of the plexiform lesion stain positive for this marker of proliferation (arrow). Endothelial cells that line normal vascular lumens in the lung are quiescent and rarely demonstrate any positive staining (original x 400).

 
The question then becomes, what could lead to this proliferative, phenotypically abnormal endothelial cell? How is this potentially genetically predisposed cell activated? The mechanisms are yet to be fully elucidated, but a number of stimuli have been identified as potential activators. Included in this list are shear stress, genetic mutations such as bone morphogenetic protein receptor II, autoimmunity (eg, CREST [calcinosis, Raynaud phenomenon, esophageal dysmotility, telangiectasia], systemic lupus erythematosus), inflammation, drugs, and viruses (Fig 3 ). Because our group studies shear stress and the association of severe pulmonary hypertension and viral infections, the remainder of the discussion will focus on these two factors as potential activators of disease.



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  		Figure 3. Potential mechanistic factors in the activation of a susceptible endothelial cell to become proliferative and dysfunctional. Hep = hepatitis; BMPR = bone morphogenetic protein receptor; ds = disease; SLE = systemic lupus erythematosus; CREST = calcinosis, Raynaud phenomenon, esophageal dysmotility, telangiectasia.

 

   Severe Angioproliferative Pulmonary Hypertension and Viral Infections
TOP
 Introduction
 Severe Angioproliferative...
Severe Pulmonary Hypertension...
 References
 
Three viruses have been studied in the development of severe pulmonary hypertension: HIV, hepatitis B, and human herpes virus (HHV)-8 (also known as Kaposi sarcoma herpesvirus). There is a clear association between HIV infection and the development of severe pulmonary hypertension.26 However, this virus has not been found in the lungs of patients with severe pulmonary hypertension associated with HIV, thus arguing for an indirect role, perhaps via production of cytokines and growth factors. Genetically predisposed individuals may have abnormal growth factor signaling in response to a viral infection.

Pulmonary hypertension is also a well-recognized complication of chronic liver disease.2728 Whether this risk factor is due to portal hypertension, viral infection, or a combination of both remains to be determined. There are reports to suggest that hepatitis B virus activates angiogenesis via stabilization of hypoxia-inducible factor-1{alpha}.29 Hypoxia-inducible factor-1{alpha} mediates transcription of the VEGF gene. Its overexpression is associated with tumor angiogenesis and proliferation.30

We have demonstrated the protein and genes of the vasculotropic Kaposi sarcoma virus, HHV-8, in the lungs from patients with primary, but not secondary, pulmonary hypertension.31 HHV-8 is a recently discovered {gamma}-herpes virus that causes classical, endemic, and HIV-1–associated Kaposi sarcoma; HIV-1 associated B-cell primary effusion lymphoma; and approximately 50% of Castleman disease.323334 Human endothelial cells infected with Kaposi sarcoma-associated herpesvirus G protein-coupled receptor change their phenotype and become immortal.35 This finding is important because HHV-8 infection of endothelial cells in the lungs of patients with severe pulmonary hypertension could lead to a monoclonal proliferation of cells in plexiform lesions and could also pave the way for somatic mutations. However, the lack of HHV-8 infection in one third of the primary and all of the secondary forms of severe pulmonary hypertension suggests that other viruses or endothelial cell phenotype-altering mechanisms must be considered in the sequence of events that leads to plexiform lesion production.


   Severe Pulmonary Hypertension and Shear Stress
TOP
 Introduction
 Severe Angioproliferative...
 Severe Pulmonary Hypertension...
References
 
Plexiform lesions most commonly form just distal to bifurcation sites of small pulmonary arteries (approximately 50 to 300 µm).24 Why are these endothelial cell proliferations found specifically at these sites in the lung? Possible explanations include the following: (1) the fluid-dynamic environment distal to the bifurcation causes endothelial damage, which is followed by a proliferative reaction; (2) the cells present at these locations are different from the endothelial cells found elsewhere in the lung or other organs; and (3) a combination of the two.

How does one study the fluid dynamics of these lesions? The difficulty in studying these lesions lies partly in the small size of the vessels; it is not possible to directly measure the shear stress directed against the endothelial lining. Additionally, blood is not water; it is a non-Newtonian fluid and exhibits a number of anomalies, including shear thinning of viscosity and anisotropy (the properties of the fluid are different in the direction of flow). Using computational fluid dynamics, however, one can perform complex computer simulations of various flow conditions at vessel branch points. These methods are widely used in various industries and have been well validated with comparisons to experimental modalities.

We used the stochastic rotation dynamics method to calculate the fluid dynamics of a bifurcating blood vessel. This mesoscopic model efficiently incorporates multiparticle collisions within a complex geometry. The vessel model was taken from a bifurcating small pulmonary artery from a normal human lung (Fig 4 , top left, A). A computerized polygon mesh, composed of 3,811 facets, was created. The simulation used 1 million fluid particles with 45,000 RBCs. The results (Fig 4, top right, B) demonstrated that shear forces maximize both proximal to and distal from the bifurcation, with a relative sparing of the immediate bifurcation site. The high shear forces distal to the bifurcation site are analogous to the location of plexiform lesions in severe pulmonary hypertension (Fig 4, bottom, C). Additional flow calculations reveal that flow remains entirely laminar at physiologic velocities and that turbulence is only seen when flows are 100 times greater than what would be considered physiologic.



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  		Figure 4. Top left, A. Stained section of a bifurcating small pulmonary artery used for construction of the computerized polygon mesh (hematoxylin-eosin, originalx100). *Indicates site of bifurcation. Top right, B: Three-dimensional computerized version of vessel in top left, A. Sites of high shear stress are indicated in red. Low shear stress areas are in blue. Note that the high shear stress locations occur upstream of the branch and distal to the bifurcation. The bifurcation site itself is in an area of low shear stress. *Indicates site of bifurcation ~100x. Bottom, C: Plexiform lesions (arrows) occurring just distal to the site of bifurcation (*) in a patient with severe pulmonary hypertension (hematoxylin-eosin, originalx100).

 
If these sites of high shear stress correspond to the sites of plexiform lesions, then does shear stress itself cause endothelial cell proliferation? Using an artificial capillary system, we have shown that endothelial cells exposed to high shear stress for up to 3 weeks proliferate and occlude the intraluminal space at a much greater rate than do endothelial cells exposed to low shear stress for 3 weeks. Sakao et al36 have also demonstrated in the artificial capillary system that shear stress causes apoptosis and subsequent proliferation of endothelial cells. It is possible that initial shear stress-induced apoptosis determines the degree of subsequent cell proliferation. Interestingly, if exogenous recombinant vascular endothelial growth factor (rVEGF165) is added to the same system under the same conditions, there is a preponderance of monolayer formation in both the low and high shear stress systems (Fig 5 ), arguing for a possible endothelial monolayer maintenance role for VEGF in the pulmonary vasculature. Using the artificial capillary system, we also show that PPAR-{gamma} is decreased in endothelial cells exposed to shear stress for 3 weeks.4 This is consistent with the loss of PPAR-{gamma} described in plexiform lesions and, by microarray gene expression screening, the decrease of PPAR-{gamma} gene transcripts in lung tissue samples from patients with severe pulmonary hypertension.



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  		Figure 5. Endothelial cells grown in artificial capillary tubes for 3 weeks under conditions of high shear stress (analogous to systemic artery pressures) demonstrate marked proliferation (> 50% of tubes occluded by intraluminal proliferations of cells). Parallel tubes seeded with the same number of endothelial cells, but grown under conditions of low shear stress (analogous to normal pulmonary artery pressures) show a marked decrease in tube occlusion. Adding rVEGF165 to the system decreased the number of occluded tubes by approximately half.

 
Although the artificial capillary system has provided us preliminary data on the effects of shear stress on endothelial cells in a long-term, pulsatile flow system, the ideal system is one in which a bifurcating artificial vascular structure can be seeded with endothelial cells and studied using pulsatile flow. In collaboration with microfluidic researchers at the Colorado School of Mines, fluid channels in a clear acrylic substrate have been fabricated using a lithographic process (Fig 6 ). By growing endothelial cells in this tissue culture model under pulsatile fluid flow, we can examine regions of endothelial cell growth and correlate the results with the predicted shear stresses from the fluid dynamic simulation. This model may ultimately help us answer important mechanistic questions as to potential initiating and amplifying factors in the pathogenesis of severe, plexiform pulmonary hypertension.



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  		Figure 6. Bifurcating fluid channels in an acrylic substrate allow for endothelial cell seeding and recirculating pulsatile flow. The flow enters through the center of the acrylic vascular model and exits at three ports at the perimeter. The system is translucent, allowing for continuous monitoring of endothelial cell growth.

 


   Footnotes
 
Abbreviations: HHV = human herpes virus; PPAR = peroxisome proliferator-activated receptor; rVEGF165 = recombinant vascular endothelial growth factor; VEGF = vascular endothelial growth factor


   References
TOP
 Introduction
 Severe Angioproliferative...
 Severe Pulmonary Hypertension...
 References
 

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