Vascular involvement in systemic sclerosis (SSc)
M.B. Kahaleh
M. Bashar Kahaleh, M.D., Professor of
Medicine and Chief, Division of Rheuma-
tology, Department of Medicine, Medical
College of Ohio, 3120 Glendale Avenue,
Toledo, Ohio 43614-5809, USA.
Clin Exp Rheumatol 2004; 22 (Suppl. 33):
S19-S23.
Copyright CLINICAL AND EXPERIMENTAL
RHEUMATOLOGY 2004.
Key words: Endothelial cells, endo-
thelin-1, nitric oxide, cytomegalovirus
(CMV), circulating endothelial cells,
apoptotic endothelial cells.
ABSTRACT
The vascular endothelium came into
view almost three decades ago follow-
ing the introduction of the vascular hy-
pothesis in scleroderma pathogenesis
by Dr. LeRoy. Since that time, the endo-
thelial cells, and other vascular cells,
became the focus of investigations aim-
ed at eludicating the etiology, patho-
genesis and treatment of scleroderma.
This review will summarize Dr. LeRoy's
commitment to the disease, the relevant
progress made since the introduction of
the vascular hypothesis, and what we
have learned since then about the vas-
cular disease in scleroderma.
Introduction
In 1976 I joined Dr. LeRoy’s section as
a first-year rheumatology fellow. A few
months into my fellowship and upon
his return from a Gordon conference on
cell biology, Dr. Leroy suggested that I
should explore techniques to isolate
and investigate vascular endothelial
cells in vitro. He affirmed that this cell
type would be the cell type of the de-
cade – and it has been the cell type of
that decade and near the top of impor-
tant cell types ever since. He talked
about a presentation in that conference
given by Dr. E Jaffe (1), who introduc-
ed the first technique to isolate endo-
thelial cells from umbilical cord veins.
This assignment represented an oppor-
tunity to break away from the hectic
clinical work that involved patient’s in-
terviews, physical examinations, data
gathering, and the dreadful correspon-
dence that must be sound scientifically,
clinically and most importantly linguis-
tically. Three decades later, I am still
working on techniques to isolate endo-
thelial cells, but now from skin biop-
sies, from peripheral blood, or from
bone marrows. This early interaction
impacted my professional career and is
an illustration of the profound influ-
ence that Dr. LeRoy had on his associ-
ates.
Dr. LeRoy’s interest in the role of vas-
S-19
cular endothelium in health and disease
begun early in his career. Still, it was
the introduction of the vascular hypoth-
esis in SSc pathogenesis in 1975 that
established him as the leader in the vas-
cular aspects of this disease (2). Al-
though many notable studies of vascu-
lar disease in SSc preceded the intro-
duction of the hypothesis, particularly
by Drs. Fleischmajer and Norton (3, 4),
the hypothesis helped to focus research
efforts on the biology and pathology of
endothelial cells and vascular function
in SSc. Since that time the endothelial
cell has become the focus of numerous
studies as the target of injury in the dis-
ease that initiates a cascade of events
including platelet activation and vascu-
lar dysfunction. Mechanisms leading to
endothelial dysfunction, intimal prolif-
eration and vascular wall thickness, tis-
sue hypoxia and the impact of hypoxia
on organ fibrosis have been partially
identified. Therapeutically, vascular
studies established the rational basis
for the use of angiotensin converting
enzyme inhibitors for scleroderma
renal crisis, calcium channel blockers
for peripheral vascular ischemia, and
the endothelin receptor antagonists for
pulmonary hypertension and digital
ischemia. These therapeutic break-
throughs have improved the plight of
the SSc patient although, despite the
symptomatic improvement, direct
regression of the vascular lesions has
not been attained.
Current thinking on SSc vascular dis-
ease suggests an autoimmune contribu-
tion, chemical modifications of the en-
dothelium and a possible viral trigger
as was proposed and passionately pur-
sued by Dr. LeRoy later in his career
(5). This paper will summarize some of
what we have learned in the last three
decades about scleroderma vascular
disease.
The vascular problem in
scleroderma
Prominent SSc vascular abnormalities
Vascular involvement in SSc / M.B. Kahaleh
are noted in the capillaries and the
small blood vessels (6). Swelling of the
intima and intimal proliferation with
mononuclear cell infiltration are seen
in vessels of 150 to 500 µm diameter.
In capillaries, the vascular disease is
characterized by distorted and irregular
capillary loops described in all
involved organs and half or more of the
expected capillaries are missing (by
morphometric capillary density mea-
surements), probably obliterated by
endothelial apoptosis and removal. This
underperfused state (ischemia, acido-
sis) should be fertile soil for neoangio-
genesis, but new capillaries are rare
and broad avascular areas are common,
suggesting defective pathways in
angiogenesis (7). On the ultrastructural
level, the earliest changes in the ede-
matous stage of the disease consist of
large gaps between the endothelial cells
(EC), vacuolization of EC cytoplasm
with an increase in the number of basal
lamina-like layers and occasional
entrapment of lymphocytes and cellu-
lar vesicles (8). Further signs of nuclear
injury in association with cell mem-
brane disruption occur in more
advanced cases. Perivascular cellular
infiltrates are prominent in the early
stages and consist of infiltrating
macrophages, T and B cells, with the
predominance of CD4+ T cells and γ/δ
receptor bearing T cells (9).
In the arteries, intimal proliferation of a
uniform and symmetrical nature forms
a neointima indistinguishable from that
formed in other autoimmune diseases,
in chronic homograft rejection, and in
accelerated atherosclerosis such as re-
stenosis after coronary bypass (10).
The principal cell forming the neointi-
ma is probably a smooth muscle cell
from the vessel wall media which be-
comes activated, leaves its resting ex-
tracellular matrix (ECM) microenvi-
ronment, exhibits smooth muscle actin
in its cytoskeleton, and is called a myo-
intimal cell. In SSc the neointima
seems to form in waves, as seen in re-
peatedly reduplicated internal elastic
membranes (11).
Vascular dysfunction in SSc
The earliest signs of vascular dysfunc-
tion in SSc include enhanced vascular
permeability and dysregulated control
of vascular tone; progressive reduction
of organ blood flow occurs in well-
established disease. The cellular bases
for these abnormalities are related to a
pathologic shift in the EC functional
profile from an anticoagulant, anti-in-
flammatory and vasorelaxent cell type
to a procoagulant, vasospastic and pro-
inflammatory phenotype. In SSc, an
imbalance in endothelial signals (in-
creased endothelin release and impair-
ed endothelial dependent vasodilatory
mechanism, EDRF), enhanced platelet
aggregation and deficient sensory neu-
ropeptides lead to the well-recognized
vasospastic propensity in the disease.
Defective compliance of the vessel
wall due to vessel wall fibrosis is also
involved, particularly in the advanced
stages of the disease. The impact of im-
balanced signals is best illustrated by
the imbalance between the production
of endothelin and nitric oxide (NO)
produced by eNOS.
Endothelin
The potent vasoconstrictor peptide en-
dothelin-1 (ET-1) is a true pleiotropic
cytokine that mediates vasospastic, mi-
togenic, profibrotic and inflammatory
effects. Increased plasma ET levels have
been associated with a number of va-
scular disorders, including Raynaud’s
phenomenon and SSc – particularly
diffuse SSc, SSc lung disease, pulmo-
nary hypertension and renal crisis (12,
13). Increased ET-1 expression in mi-
crovascular endothelial cells (MVEC)
of the upper dermis in association with
an increased number of ET-1 binding
sites (particularly type B receptors) is
reported in SSc skin and lungs (14).
Due to the wide-ranging biologic ef-
fects of ET it was proposed as the link
between the vascular and fibrotic pro-
cess in SSc. The recent therapeutic suc-
cesses of ET receptor antagonists in
pulmonary hypertension and digital is-
chemia support the important role of
ET in vascular disease; however, the
link with fibrosis still need to be exam-
ined by direct evaluation of the an-
tagonists on the fibrotic burden in SSc.
A positive effect on fibrosis will vali-
date the vascular hypothesis as envi-
sioned by Dr. LeRoy. Another area that
S-20
still needs examination is the nature of
ET stimulus in SSc. The enhanced pro-
duction may be related to central alter-
ation in endothelial cells leading to
defects in the feedback loops that is
normally seen in the healthy state.
Endothelial Dependent Relaxation
Factor and nitric oxide
Deficient endothelial dependent relaxa-
tion in SSc is suggested by impaired
maximal responses to endothelial de-
pendent vasodilators with normal re-
sponses to endothelial independent di-
lators (15, 16) and by reduced expres-
sion of eNOS in the skin and microvas-
cular EC in vitro.
The consequences of impaired endo-
thelial nitric oxide (NO) release in SSc
are not only related to defects in vascu-
lar tone control but may also influence
other pathological events that can pro-
mote and augment disease pathogene-
sis. NO is known to inhibit platelet ag-
gregation, and adhesion molecule ex-
pression (17-19). Furthermore, NO is a
powerful inhibitor of smooth muscle
cell proliferation and acts as a potent
chemical barrier to oxidation injury
(20). Thus, a reduction in steady state
endothelial NO production may pro-
mote vascular wall inflammation, inti-
mal proliferation and platelet aggrega-
tion, and augment oxidation injury. A
fine balance between NO and ET exists
in the healthy state where ET triggers
NO release upon binding to the type B-
ET receptors on the endothelium. Re-
leased NO in turn suppresses ET re-
lease, restoring the vasodilatory status
of the blood vessels. Several experi-
mental data suggest a disruption of this
feedback loop, resulting in sustained
ET production in SSc. Further under-
standing of this process and pondered
therapeutic intervention may help in
eliminating the progressive obliterative
vascular disease in SSc and allow the
ultimate testing of the vascular hypoth-
esis.
The nature of endothelial injury
Recent studies identified EC apoptosis
as an early event in SSc pathogenesis.
EC apoptosis was first noted in chick-
ens from University of California at
Davis lines 200/206 that develop he-
Vascular involvement in SSc / M.B. Kahaleh
reditary systemic connective tissue di-
sease resembling SSc (21). In the human
disease, EC apoptosis is seen mainly in
association with the early inflamma-
tory stage of the disease, suggesting a
causal association. EC apoptosis is not
unique to SSc since it is abundant in
atherosclerotic vascular lesions, organ
rejection, TTP, the hemolytic uremic
syndrome and other vascular disorders
(22, 23). The mechanism of EC apopto-
sis in SSc is not known. The suggested
mediators of EC injury in SSc include
products of cytotoxic T cells, antibody
dependent cellular cytotoxicity, anti-
endothelial antibodies, and ischemia
reperfusion with oxidant injury.
Involvement of cytotoxic T cells is more
plausible as a trigger for EC apoptosis
in view of the histologic and exper-
imental findings in the disease. It is
known that EC apoptosis results from
EC interaction with cytotoxic T cells
either by fas or granzyme/perforin-
related mechanisms (24). The CD4+ T
cells induce EC apoptosis by a fas-
related mechanism – as seen in the cy-
tolytic T cell killing of vascular endo-
thelium in the rejection reaction, where
CD4+ cytolytic T cells are the most
prominent effector cells and where all
apoptotic EC express the fas ligand
(25). The granzyme/perforin system
mediates EC apoptosis by the major
cytotoxic cells – the CD8+ T cells, NK
and LAK cells. Granzymes gain access
to the cells following cellular mem-
brane damage by perforin. The mecha-
nism whereby granzymes induce EC
apoptosis, although not well document-
ed, is fas independent (25). Viral in-
fection of the endothelium may also
trigger cytotoxic EC apoptosis when
EC cells express viral antigen. What is
intriguing here is the fact that certain
viral infections of the vascular endo-
thelium prime cytolytic T cells to kill
non-infected innocent bystander endo-
thelial cells, as seen in cytomegalovirus
(CMV) viral infection (26).
Possible microbial triggers
A persuasive and credible argument
supporting a viral trigger of SSc was en-
thusiastically presented by Dr. LeRoy
because of his observation of increased
levels of antibodies to CMV, a large
Circulating EC
Control
Fig. 1. Circulating anuclear endothelial cells in 8 SSc patients and 7 matched control subjects. The
mean number of CEC in the controls was 3.01 ± 0.7 and in SSc patients 5.5.1 ± 1.6 (mean ± SD, P <
0.002).
DNA herpes virus in SSc patients, and
because accelerated neointima forma-
tion had already been indirectly associ-
ated with CMV in graft rejection and
arterial restenosis (27). Moreover, what
is known about CMV infection fits well
into the scenario of SSc vascular patho-
genesis. CMV-infected endothelial cells
round up, detach, and circulate to dis-
tant capillary beds, which serve to dis-
seminate the virus and infect distant
EC, leading to diffuse systemic vascu-
lar disease. This process could also lead
to endothelial cell apoptosis, as well as
subsequent autoimmunity, including
anti-endothelial cell and antiphospho-
lipid antibodies. Dr. LeRoy theorized
the mechanism of a latent CMV infec-
tion in which immediate early (IE1 and
2) CMV genes drive host gene expres-
sion. SSc skin lesions have been shown
to contain mononuclear cells which
express TGFβ1 alongside interstitial fi-
broblasts which express α1(I) collagen
mRNA. These cells would also be ex-
pected to overexpress ICAM-1 and IL-
6, characteristic abnormalities of the
activated scleroderma fibroblast (a my-
ofibroblast), which are also induced by
CMV IE1-IE2. Therefore many of the
pathological features of SSc are con-
sistent with a latent CMV infection
where CMV promoter genes drive host
cytokine and matrix gene expression
S-21
SSc
leading to neointima, capillary oblitera-
tion and interstitial fibrosis. In vitro
CMV induces TGFβ1 (and thereby se-
veral collagen genes) and adhesion mo-
lecules, among its many other attribu-
tes. For SSc pathogenesis a latent CMV
infection is a very interesting hypothe-
sis that should be vigorously examined.
Circulating markers of the vascular
disease
One of the intermediate goals of the va-
scular hypothesis was to identify mark-
ers of vascular dysfunction. Monitoring
these markers would help in short-term
trials to evaluate the effectiveness of
therapies on the vascular disorder.
Many markers were identified over the
years including von Willebrand Factor
(vWF), angiotensin converting enzy-
me, β-thromboglobulin and platelet
factor VI, soluble adhesion molecules,
thrombomodulin and many others.
Many of these markers positively cor-
related with the extent of visceral in-
volvement, disease prognosis, active
phase of the disease and certain serolo-
gies. One of the markers that was exa-
mined early but not reported was circu-
lating endothelial cells.
Circulating apoptotic endothelial cells
(CAEC) in SSc
Damaged endothelial cells may slough
Vascular involvement in SSc / M.B. Kahaleh
off the vascular wall and circulate as
the anuclear remains of the cell skele-
ton that are increased in number in vas-
cular diseases. The method to evaluate
CAEC is based on collecting platelet-
rich plasma, aggregating the platelets
with thrombin, and counting the cell
carcasses in the platelet-poor plasma
following centrifugation (28). We eval-
uated the CAEC in 8 SSc and 7 match-
ed control subjects. The number of cir-
culating cells was higher in SSc pa-
tients (Fig. 1). Moreover, the circula-
ting numbers increased dramatically in
patients undergoing interferon-γ thera-
py in association with digital infarcts.
These observations were not published
because of the uncertainty of the origin
of the cells. Still, the presence of non-
hematological cells in the peripheral
circulation in patients with cancer was
reported in 1934 (29). Recent advances
in cell isolation and immunohistoche-
mical examination revealed that some
of the circulating cells are endothelial
in origin (30). Increased numbers of cir-
culating endothelial cells (CEC) have
been described in several pathologic dis-
orders that have in common the pres-
ence of vascular injury. Thus, increased
CEC numbers were described in sickle
cell anemia (31), rickettsial (32) and cy-
tomegalovirus (CMV) infections (33),
thrombotic thrombopenic purpura (TTP,
34), acute coronary syndromes (35),
Behçet’s disease (36), vasculitis (37)
and in patients with active SLE (38).
Normal adults have 2.6 ± 1.6 CEC per
milliliter of peripheral blood (31). This
low number reflects the exceptionally
slow turnover rate of vessel wall endo-
thelium in the healthy endothelium (39).
At least half of the CEC are derived
from the microvasculature as defined
by CD36 positivity (31). The phenotype
of these cells is postulated to reflect the
status of the endothelium in situ (40,
41), but whether the CEC themselves
are derived from vessel walls is not
known. Nonetheless, the measurement
of CEC provides direct evidence of en-
dothelial injury and should be exam-
ined in SSc.
Until recently, it was generally thought
that the formation of new vessels in
adults occurred exclusively through the
extension of mature existing blood ves-
sels and the associated vascular endo-
thelium. A growing body of evidence
now suggests that bone marrow-deriv-
ed endothelial progenitor cells (EPCs)
circulate in the blood and, at least in
animal models, can play an important
part in the formation of new blood ves-
sels in these pathologic conditions (42,
43). Laboratory evidence suggests that
these cells express a number of endo-
thelial-specific cell surface markers and
exhibit numerous endothelial proper-
ties (42, 43). In addition, when these
cells are injected into ischemic animal
models, they are apidly incorporated
into sites of neovascularization (44-
46). These studies suggest a potential
use of progenitor cells for improve-
ment of therapeutic vasculogenesis in
patients with a variety of vascular dis-
eases. Attempts to characterize and
propagate the progenitors cells in SSc
are ongoing.
Conclusion
The etiology and pathogenesis of SSc
remain unknown. Nonetheless, signs of
vascular injury and devascularization
of involved organs in association with
evidence of profound endothelial dys-
function are well documented. Fibrob-
last activation leading to tissue fibrosis
and immune involvement constitute the
two other fundamental pathologic pro-
cesses in the disease in addition to the
vascular disorder. Countless central
issues in the pathogenic process of SSc
remain poorly understood. Issues relat-
ed to the initial trigger in the disease,
the nature of immune activation, mech-
anisms of intimal proliferation and the
relationship of vascular injury to tissue
fibrosis are some of the unresolved
essential questions.
The fact that the vascular tree, particu-
larly the microcirculation, is the target
tissue in this disease is now well estab-
lished. The destruction of microvessels
leading to the clinically recognized
state of chronic organ ischemia and tis-
sue underperfusion in SSc is funda-
mental to its pathogenesis. Chemical
modification of the endothelium, reper-
fusion injury by mechanisms of RP, or
viral infection of EC are some of the
proposed triggers. The microvascular
damage in the disease, which includes
S-22
EC apoptosis, may not be related to the
direct effect of virus replication but
may be linked to immune mediated en-
dothelial injury triggered by viral in-
fection. Knowledge of the immune me-
chanisms in disease pathogenesis may
offer an opportunity to develop a multi-
ple step strategy for therapeutic inter-
vention. The introduction of the vascu-
lar hypothesis by Dr. LeRoy ushered in
a most exciting era in SSc investiga-
tion, the fruits of which we are already
enjoying now in the care of SSc pa-
tients. The determination to pursue the
vascular hypothesis will undoubtedly
be a productive pathway of research in
SSc for some time to come.
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