|
 Norbert
F. Voelkel, MD, Laima Taraseviciene-Stewart, and Carlyne
Cool, MD
Pulmonary Hypertension
Center and Department of Pathology
University of Colorado Health Sciences Center
Denver, Colorado
ANALYSIS OF LUNG TISSUE IN SEVERE
PULMONARY HYPERTENSION HAS CHANGED CONCEPTS OF PATHOGENESIS
The rather recent development of specific antibodies
and molecular probes allows the identification, localization,
and colocalization of growth factor genes and proteins,
transcription factors, enzymes, oncogenes, and markers
of cell growth and cell death.1-5
Standard hematoxyline/eosine staining continues to be
used for routine screening of abnormalities of the lung
tissue, but special stains permit answers to specific
questions. Lung tissue extracts from patients with pulmonary
hypertension (PH) have also been used for Western blot
protein analysis3, 6
or for mass spectroscopy analysis of lipid mediators7
and for RNA and DNA work;6, 8,
9 quantitative PCR allows the detection
of low levels of expressed genes,6,
8 and microarray analysis can establish
a signature gene expression profile that can distinguish
different forms of severe PH.10
Some investigators observed the emergence of tissue-based
information with skepticism because they believed the
data related only to end stage disease and scar tissue.
This is clearly not the case, since the tissue displays
the full spectrum of early and late lesions and different
degrees of inflammation, and contains relatively uninvolved
areas.
Which Vascular Lesions? Which
Cells?
Controversies still exist regarding the importance of
the complex vascular lesions, which include the so-called
plexiform (or glomeruloid) lesions, concentric intima
fibrosis (or onion skin lesions in scleroderma), and angiomatoid
lesions.11 Are these lesions
hemodynamically important or just markers of severe PH?
Some patients seem to have more of these lesions than
others, yet the "pruning" of the pulmonary arteriolar
tree, which can be documented radiographically and is
seen in all patients with severe PH, can be explained
as a drop out or loss of precapillary resistance vessels
by an unknown mechanism or by angiogenic lumen obliteration.
In the peripheral subpleural regions of the lungs the
vascular pruning is most evident and complex, and lumen-obliterating
lesions are likely detected here.
Muscularization of the arterioles alone is (in the opinion
of these authors) not sufficient to explain severe and
progressive PH. IN the aggregate, a number of recent studies
point to an angioproliferative process with a preference
for particular sites of the vascular tree, namely bifurcations
of precapillary arterioles, which makes severe PH a group
of diseases characterized by structural alterations of
microscopically small vessels. These alterations do not
span a great length of an individual vessel; instead,
three-dimensional reconstruction analysis shows there
is a patent lumen proximal and distal to a "plug."12
Careful examination of many serial sections of many subpleural
lung tissue samples would likely result in the detection
of many regionally obliterated vessels and lead to the
conclusion that the occlusion or partial blockage of literally
millions of these peripheral microvessels can explain
why the pulmonary artery pressure is so high (usually
50 mm Hg), and why at the time of the first hemodynamic
study only 20 to 25 percent of patients with severe PH
display a significant acute vasodilator response.
Many of our earlier pathogenetic concepts were based
on physiologic parameters, pressure, flow, and vasoconstriction
causing an increase in precapillary vascular resistance.
Understandably our though models were those of 19th and
20th century physiology, and the still fascinating acute
hypoxia-induced pulmonary vasoconstriction13
or chronic hypoxic pulmonary hypertension14,
15 animal experiments provided
tools to study pulmonary vascular tone regulation and
pulmonary vascular remodeling.16
Yet these models infringe on the human condition of severe
angioproliferative PH (SAPH) only peripherally, and it
is interesting that the development of the first endothelin
receptor blocker for clinical use in "primary"
PH was based to a large degree on the positive results
of preclinical studies in hypoxia and monocrotaline rat
models.17, 18
The assumption clearly was that these rodent models bore
enough resemblance to human SAPH, and further, that endothelin
was not only "bad" but also a central and very
critical actor in a drama played out by a large score
of characters. Because human SAPH occurs only in individuals
with a still ill-defined genetic predisposition (or susceptibility)
and because the pulmonary vascular biology and pathobiology
are most certainly very complex, these assumptions are
likely wrong or at least too simple.
New Concept of Severe Angioproliferative
Pulmonary Hypertension
Histologically all the layers of the pulmonary arterioles
- intima, media, and adventitia - and a great number of
cell types (including lymphocytes, myofibroblasts, macrophages,
mast cells, and bombesin-positive cells) are involved
and present in SAPH. The vascular lesions in SAPH are
truly complex and it is now clear that they contain not
only normal but also very abnormal, phenotypically altered
cells. Why is this important? We suggest that it is the
nature of these phenotypically altered, apoptosis-resistant
cells2, 6, 19
that sets human SAPH apart from most of the rodent models
and makes human angioproliferative PH so difficult to
treat.
A.
Plexiform lesion from a patient with severe angioproliferative
pulmonary hypertension. The nuclei of the phenotypically
altered cells in the plexiform lesion demonstrate
punctuate, brown staining (arrows) with antibodies
directed against latency associated nuclear antigen-1
(LANA-1), which is constituitively expressed in lytic
and latent human herpes virus 8 (HHV-8) infection.
LANA-1 immunohistochemical stain, 1000X. B.
H&E stain of plexiform lesion demonstrating the
characteristic exuberant endothelial cell proliferation.
A disordered arrangement of plump, spindle-shaped
endothelial cells line the multiple slit-like lumens
(*). 400X. C. Plexiform lesion co-stained
with Factor VIII-related antigen (FVIII-r.ag - an
endothelial cell marker) and a-smooth muscle actin
(a-SMA), followed by Alexa Fluor labeled secondary
antibodies. The slides are coverslipped with fluorescent
mounting media containing DAPI. Factor VIII-r.ag positive
cells fluoresce green (FITC), the cell nuclei fluoresce
blue (DAPI), and the smooth muscle fluoresces red
(rhodamine). The transitional cells, defined by their
coexpression of Factor VIII-r.ag and a-SMA, fluoresce
yellow to orange (arrows). 1000X. D. Vascular
endothelial growth factor (VEGF) immunostaining of
a plexiform lesion. Some of the cells of the plexiform
lesion show a high level of expression for this angiogenic
growth factor (arrows), while the surrounding lung
tissue is negative. 400X. |
A growing number of reports have provided evidence for
clonal cell growth and somatic endothelial cell mutations
as well as for loss of tumor suppressor genes and expression
of proteins usually not found in endothelial cells, like
5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP).19-21
Most likely a number of different trigger factors can
induce programmed cell death-apoptosis of vascular endothelial
cells. We hypothesize that "exuberant endothelial
cell proliferation"2
may involve the participation of usually quiescent stem
or precursor cells and/or perhaps bone marrow-derived
hemeangioblasts. Circulating endothelial cells occur in
greater numbers in severe PH;22
whether these cells have been sheared off in the lung
circulation or originate from other sites is unclear.
Regardless, in theory, precursor cells or phenotypically
altered endothelial cells could - perhaps through shunts
- gain access to the systemic circulation and also recirculate
into the lung.
Peripheral Blood Cell Analysis
in Diagnosis of Severe Pulmonary Hypertension
Patients with severe PH have elevated plasma levels of
norepinephrine, vascular endothelial growth factor (VEGF),
IL-1, IL-6, and endothelin;.23-25
elevated levels of serotonin have also been described.26
This altered cytokine and growth factor milieu of the
peripheral blood cells is bound to affect the gene expression
pattern and behavior of these cells.
Bull el at27 recently acted
on this concept and performed microarray gene expression
analysis of peripheral blood monocytes (PBMC) in patients
with severe PH and found that the patients' PBMC gene
expression was clearly different from that of normal PBMCs.
In addition, they identified a small number of genes that
were differently expressed when patients with so-called
primary PH and patients with secondary forms of severe
PH were compared. It is likely that analysis of PBMCs
will allow subgroup identification and possibly prediction
of treatment response. We have entered only the first
phase of this exploration.
Severe Angioproliferative Pulmonary
Hypertension and Viral Infection
The first recognized association of viral infection and
severe PH, a disease histologically indistinguishable
fro so-called primary PH, was the association with human
immunodeficiency virus (HIV) infection.28
This association and the recently recognized second association
with human herpes virus 8 (HHV-8, or Kaposi's sarcoma
virus) infection,29 raise
many new questions. These questions when answered will
lead to a more complete understanding of the pathobiology
of SAPH.
The first question is whether there is only an association
with certain viral infections - based on an altered immune
system 0 or whether the virus causes severe PH
because of death of some endothelial cells and"transformation"
of surviving cells as in Kaposi's sarcoma. Next, are there
other viruses that need to be discovered? What is the
mode of infection in HHV-8-associated severe PH? Does
severe PH also occur in virally infected primates? Is
the occurrence of severe PH in splenectomized patients30
also associated with viral infections, etc?
Infection, Inflammation, Phenotypically
Altered Pulmonary Vascular Cells, and Vasodilator Therapy
As we31 and hopefully others,
perhaps with a measure of reluctance, accept this new
pulmonary vascular pathobiology, our patients expect and
deserve new treatment strategies. These will likely not
result in "complete molecular repair" of all
of the lung lesions or a molecular remission, but in a
partial desobliteration of precapillary arterioles because
of removal of lumen obliterating cells (perhaps by induction
of apoptosis in apoptosis-resistant cells). There is enough
collective experience to say that the drugs currently
in use - all vasodilators - do not reopen occluded vessels.32
It is not clear whether antiangioproliferative agents
will be able to do the job. They may prevent further progression
because of inhibition of further angioproliferation, but
they may not remove already established lesions.
A rodent model of severe PH (mean pulmonary artery pressure
>50 mm Hg) due to lumen obliteration of precapillary
arterioles has been established.33
Inhibition of apoptosis with a broad-spectrum caspase
inhibitor prevents the development of severe PH in these
animals, but reversal of established SVP in this model
is as difficult as in the human SAPH group.34
Patients with SAPH have millions of obliterating lesions
and may not benefit from antiviral drugs. Instead, proteins
expressed only in abnormal apoptosis-resistant vascular
cells, not in normal endothelial and smooth muscle cells,
are drug targets and cell-specific homing strategies can
be developed.
Outlook
The continuous infusion of prostacyclin was the first
real breakthrough in the treatment of severe PH,32,
35, 36 and this
treatment has improved the survival of many patients afflicted
with this rare and deadly disease. To accept that angioproliferative
obliteration in severe PH is both pathobiologically and
hemodynamically important is the first step. We hope that
many investigators will be able to take this step. The
next step is to take the fact seriously that the lumen-obliterating
cells in SAPH are abnormal cells and actually cancerlike.19,
29 Primary PH was once called "the
cardiologist's cancer" (Greg Eliot). The removal
of these quasimalignant cells from established vascular
lesions is our new treatment goal.34
If successful, such treatment would shorten the transplantation
list.
Acknowledgement
This work has been supported by an NIH Program Project
Grant, PO1 HL 66254.
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