The Big Idea: iron-dependent inflammation in venous disease and
proposed parallels in multiple sclerosis
Professor Paolo ZamboniJ R
Soc Med 2006;99:09-07.1-5
This article is based on a lecture presented at the Tripartite
Meeting, EVF-AVF-UKVF, Royal Society of Medicine, London,
1July 2006.
IRON DEPENDENT INFLAMMATION IN CVD
Impaired venous drainage of the lower extremities, mainly due to
venous reflux or to venous outflow obstruction,leads to a cascade of
pathological events clinically graded by the clinical class (C) of the
CEAP classification (Clinical,aEtiological, Anatomical,
Pathophysiological) of chronic venous disease (CVD).
Varicose veins are the most frequent clinical sign in class C2. When
oedema complicates varicose veins, the clinical picture is graded as
C3. Pigmentation, lipodermatosclerosis and other skin changes are
classified as C4. A small but significant number of the affected
patients develop venous ulcers. Healed ulcers are classified as C5,
whereas active ulcers are C6.Altered venous haemodynamics are a
necessary but not exclusive element for explaining progression along
the
clinical classes to the point of skin lesion.
In 1982, Browseand Burnand  observed a peri-capillary fibrin
deposition and speculated that cuffs act as a barrier to oxygen
diffusion and nutrients, resulting in epidermal cell death. This
mechanism of tissue injury has not yet been demonstrated. The fibrin
cuff may be more properly considered a scaffold for tissue reparative
processes. The cuff contains fibrin, but also laminin, fibronectin,
tenascin, and types I and III collagen,encircling the dilated
capillary vein . The decline of the fibrin cuff theory over the last
twenty years has led to investigation of other factors emphasizing
inflammatory mechanisms as amplifiers of the insufficient venous
drainage. Recent studies demonstrate a pivotal role for tissue iron
accumulation in inducing and maintaining inflammation in CVD. Iron
deposits in CVD cause readily visible brownish dermal areas which
sometimes precede, but always surround, ulcers. The origin of
increased leg iron stores is extravasation of red blood cells
(erythrocytes) in conditions of significant venous stasis.
Erythrocytes are degraded by the interstitial macrophages, with the
released iron incorporated into ferritin. Over time, with increasing
overload of iron, the structure of ferritin changes to haemosiderin.
In 1988, Ackermann found a twenty-fold higher average concentration of
iron in lower limbs affected by venous ulcers as compared to the upper
arm of the same subjects.
The phenomenon of leg haemosiderin deposits seems to be significant
for the entire body, since this protein has been demonstrated in the
urine of patients affected byCVD.  Increased iron stores and
interstitial protein extravasation are potent
chemo-attractants and presumably represent the initial underlying
chronic inflammatory signal responsible for white blood-cells
recruitment and migration in the matrix .
In 1988, Coleridge-Smith observed leukocytes trapped in the venous
microcirculation secondary to venous hypertension. This work paved the
way to the investigation of the relationship between CVD and
inflammation.
The mechanism of white cell migration in the subcutaneous matrix was
further elucidated by studies of the expression of adhesion molecules
in a model of venous hypertension. Several studies confirmed the
expression of these molecules, including ICAM, VCAM and selectins.
Such adhesion molecules block circulating white cells on the vein wall
and facilitate transmigration into the tissue. The predominant cells
migrating into the extra-cellular matrix are macrophages and T-
lymphocytes.
Macrophages take up iron accumulated in the tissue and store it in
intracellular ferritin-like structures. Intra- and extra-cellular
overload of iron in the tissue could potentially be dangerous for
generation of free radicals due to possible release of free iron from
deposits.Wenket al.and Yeoh-Ellerton found increased iron levels in
exudates from chronic leg ulcers as compared to acute wounds.
They also observed significant concentrations of metabolites from
oxidative stress. The final step of the pathogenetic chain leading to
matrix disruption and ulcer development involves over-expression of
matrix metallo-proteases (MMPs) that are not substantially balanced by
their physiological tissue inhibitors (TIMPs). MMPs cause a substrate-
specific degradation of matrix components, including collagen, elastin
and laminin.Unrestricted MMP activity can lead to matrix break down
and ulcer onset. Some experiments demonstrate that local iron overload
may induce MMP hyper-activation through the so-called MMP iron-driven
pathway.However, the iron hypothesis does not readily explain why leg
iron deposits in CVD produce lesions only in some individuals. We
hypothesized that such individual differences could be genetically
determined, and investigated the role ofthe C282Y and H63D mutations
of the HFE gene, associated with hemochromatosis in Northern European
populations. C282Y mutation significantly increases the risk of ulcer
inprimary CVD by more than six-fold, while patients carrying the H63D
variant have an earlier age of ulcer onset by almost 10 years.5HFE
mutations are associated with increased iron efflux from the
macrophage. Our findings support the hypothesis that lesions are
promoted by enhanced iron release and ROS generation.
PARALLELS BETWEEN INFLAMMATION IN CVD AND IN MS
Clinical observations sometimes suggest alternative explanations of
previous findings. During a duplex scanning examination on the carotid
arteries of a 55 year old patient with multiple sclerosis (MS), I
observed an unexpected reflux from the chest into the internal jugular
vein after the patient coughed involuntarily. I then noted this
unusual phenomenon in other MS patients.
There were previous reports of a close relationship between dilated
cerebral veins and inflammatory lesions In MS. Fog showed that the
plaques of cerebral MS arise from definite segments of large
epiventricular veins and that the lesions digitating out into the
cerebral hemispheres also consistently evolve in a corresponding vein
relationship.
Putnam showed plaques lined with gliotic tissue containing large
veins, surrounded by hematogenous pigment. On a cerebral hemisphere
medial aspect, a number of vein-centred plaques spread beneath the
lateral ventricular wall and surge up off of the corpus callosum under-
surface.
The stem and first branches of a large ventricular vein have grooved
wide beds whose breadth is nearly three times that of the involved
vessel diameters, a detail reminiscent of Charcot's first
documentation of cerebral MS. Fibrin cuffs are not an exclusive
finding of CVD, but are commonly visible around cerebral veins in the
course of MS and today they are interpreted as ongoing reparative
processes. MRI venography confirms in vivo the close relationship
between the main cerebral veins and the inflammatory plaques. In MS
lesions, a central vein was visible. When cortical lesions occur, they
arise within the territory of the principal cortical veins. In another
study, contrast MRI allowed documentation of the break-down of the
blood-brain barrier (BBB). Such an injury preceded other MRI
abnormalities and the clinical evidence of a new lesion. This supports
the view that a defect in the BBB, and therefore inflammation, is an
early and possibly crucial event in the pathogenesis of a new lesion
in MS. Inflammation in MS is characterized by expression of adhesion
molecules, followed by a migration of macrophages and T-cells across
the BBB. Infiltration of the matrix by macrophages, as in CVD, is
considered a crucial step . In both situations,macrophages appear with
considerable intracellular iron stores due to phagocytosis of
senescent erythrocyte. Iron overload in MS plaques has been
demonstrated in vivo by MRI.  In addition, we observed haemosiderin in
the urine of patients with active inflammation of MS (personal
unpublished data).Iron-laden macrophages carrying the HFE mutation
display increased iron export, increasing the risk of generation of
free iron and free radicals, possibly extending tissue lesions. A
study from Australia 29 suggests that C282Y-HFE mutation is increased
in MS cases of North Western European origin and supports further
investigations into the role of iron metabolism in the severity of MS.
As in a venous ulcer, a key determinant of tissue injury is played by
MMP9. Exactly as in CVD, the over-expression of MMP9 is insufficiently
counter balanced by its tissue inhibitor TIMP-1. MMP9 can trigger
leukocyte transendothelial traffic through an altered BBB, and serum
active MMP9/TIMP-1 is now considered an appropriate indicator of
ongoing MS inflammation. Despite histological findings showing
haemosiderin deposits encircling the central vein of MS lesions , the
iron-MMP pathway of activation is not considered in MS literature.
DISCUSSION
Table 1 summarizes the iron-dependent inflammatory chain in CVD and
shows impressive pathophysiological similarities with MS. The critical
point of the proposed parallelism between CVD and MS involves venous
haemodynamics. In CVD, altered venous haemodynamics are considered the
trigger mechanism of subsequent inflammation. In contrast, altered
venous haemodynamics in MS are poorly studied. The possible role of
venous reflux/obstruction in cerebral and spinal veins requires
additional investigation. Earlier literature indicated that the
inflammatory lesions spread counter-current to the normal
venous flow direction, and the process of cerebral multiple sclerosis
advances in a direction diametrically opposed to that of normal venous
flow. Such circumstances should be further investigated with the help
of advanced neuro-imaging technology.Although investigations on the
role of iron in MS are still few, some evidence supports a pivotal
role for iron in MS inflammation. The effect of manipulation of iron
level was investigated in EAE, a form of induced autoimmune
encephalomyelitis in mice used as an experimental model of MS. The
incidence of EAE was 60-70% in mice with anormal iron level and in
iron-overloaded mice, but 0% in iron-deficient mice. The findings
suggest that iron deficiency provides protection from the development
of EAEand also challenge traditional views on what constitutes a
normal level of stored iron. The authors noted that, 'The failure of
iron-deficient mice to develop EAE is impressive. Many of the
pharmaceutical approaches to inhibiting EAE are less effective than
iron deficiency.'Another group investigated the serum concentration of
soluble transferrin receptor (sTFR) in a group of MS patients. The
levels were found to be significantly higher inpatients with active
MS, either in progressive or relapsing-remitting clinical form, than
in controls. Serum ferritin levels were also significantly elevated in
patients affected by the active and progressive form. Both findings
support the hypothesis above described, which proposes local iron
overload as the initial signal of the inflammatory chain in
MS.Although the primum movens of MS is still elusive, these studies
suggest that iron-dependent mechanisms of inflammation seen in CVD
could be relevant to MS. Future work on MMPs and on iron/macrophage
interactions appears especially promising. However, because of its
relevant epidemiology and its easily visualized lesions, CVD is an
ideal model for investigating iron mediated mechanisms of tissue
injury of venous and inflammatory origin, as well as the use of
deliberate induction of iron deficiency as a treatment modality.
Acknowledgments
This research was supported by the Italian Ministry for the University
and the Scientific Research and by the Foundation Cassa di Risparmio
di Ferrara.

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