The Big Idea: Iron-dependent inflammation in venous disease and proposed parallels in multiple sclerosis

J R Soc Med 2006;99:589-593
© 2006 Royal Society of Medicine


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J R Soc Med 2006;99:589-593
© 2006 The Royal Society of Medicine


Paolo Zamboni

Professor, Vascular Diseases Center, University of Ferrara, 44100
Ferrara, Italy




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).1
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, Browse and
Burnand2 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
(Figure 1A). 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

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Figure 1. Panel A, classic fibrin cuffs (arrow) thicken veins (v) in a venous
ulcer bed, 40 x. Panel B, fibrin cuffs (arrow) encircles proliferated thick
walled veins (v) in a peri-ventricular MS plaque, 30 x.
Panel A is
courtesy of Professor Caggiati, Rome, Italy. Panel B is modified from Adams
CW. A Colour Atlas of Multiple Sclerosis. London: Wolfe Med,


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
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.8 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 by


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 (Figure
). 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.10 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
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

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Figure 2. Panel A, intra and extra-cellular iron deposits (ID) encircle a dilated
vein (V) in a cerebral MS plaque, Perls’ method 150 x. Panel B, intra and
extra-cellular iron deposits (ID) encircle a dilated vein (V) in venous ulcer
bed, Perls’ method 80 x


Macrophages take up iron accumulated in the tissue and store it in
intracellular ferritin-like structures
(Figure 3B). 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
Wenk et
.7 and
found increased iron levels in exudates from chronic leg ulcers as compared to
acute wounds. They also observed significant concentrations of metabolites
from oxidative

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Figure 3. Panel A, top and bottom: iron-laden macrophages migrated in the cerebral
extra-cellular matrix in course of MS, Perls’ staining, 200 x. Panel B, top
and bottom: iron-laden macrophages migrated in the extra-cellular matrix in
course of CVD, Perls’ staining, 400 x


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
Some experiments demonstrate that local iron overload may induce MMP
hyper-activation through the so-called MMP iron-driven
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 of the C282Y and H63D mutations of the HFE gene, associated with
hemochromatosis in Northern European populations. C282Y mutation significantly
increases the risk of ulcer in primary CVD by more than
six-fold,6 while
patients carrying the H63D variant have an earlier age of ulcer onset by
almost 10 years.5
HFE 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



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 (Figure 4).

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Figure 4. Reflux documented in an internal jugular vein under Valsalva in a
patient affected by MS. Inversion of the flow wave from positive to negative
(i.e. from chest to the head) is well apparent.


There were previous reports of a close relationship between dilated
cerebral veins and inflammatory lesions In MS.
Fog17 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.
Putnam18 showed
plaques lined with gliotic tissue containing large veins, surrounded by
hematogenous pigment (Figure
). 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

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
(Figure 1B). MRI venography
confirms in vivo the close relationship between the main cerebral veins and
the inflammatory plaques. In 94/95 MS lesions, a central vein was
visible.22 When
cortical lesions occur, they arise within the territory of the principal
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

Inflammation in MS is characterized by expression of adhesion
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
(Figure 3A and B). 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.28 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
A study from
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 severityof 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
counterbalanced by its tissue inhibitor TIMP-1. MMP9 can trigger leukocyte
transen-dothelial traffic through an altered BBB, and serum active MMP9/TIMP-1
is now considered an appropriate indicator of ongoing MS
Despite histological findings showing haemosiderin deposits encircling the
central vein of MS lesions (Figure
), the iron-MMP pathway of activation is not considered in MS


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
Such circumstances should be further investigated with the help of advanced

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Table 1. Common findings of the inflammatory chain in CVD and MS


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.31 The incidence
of EAE was 60-70% in mice with a normal 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
EAE31 and also
challenge traditional views on what constitutes a normal level of stored
iron.14 The
authors31 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 group32
investigated the serum concentration of soluble transferrin receptor (sTFR) in
a group of MS patients. The levels were found to be significantly higher in
patients 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.32 Both
findings support the hypothesis above described, which proposes local iron
overload as the initial signal of the inflammatorychain 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


This article is based on a lecture presented at the Tripartite Meeting,
EVF-AVF-UKVF, Royal Society of Medicine, London, 1 July 2006.

Acknowledgments This research was supported by the Italian
Ministry for the University and the Scientific Research and by the Foundation
Cassadi Risparmio di Ferrara.

Competing interests None declared.


  • Eklof B, Rutherford RB, Bergan JJ, et al. for the American
    Venous Forum International Ad Hoc Committee for Revision of the CEAP
    Classification. Revision of the CEAP classification for chronic venous
    disorders: consensus statement. J Vasc Surg2004; 40:1248
  • Browse NL, Burnand KG. The cause of venous ulceration.
    Lancet1982; 2:243
  • Joshi A, Sloan P. Role of `fibrin’ cuffs in chronic nonspecific
    oral ulceration. Wound Repair and Regeneration2004; 12:18[Medline]
  • Zamboni P, Scapoli G, Lanzara V, et al. Serum iron and
    MMP-9 variations in limbs affected by chronic venous disease and venous leg
    ulcers. Dermatol Surg2005; 31:644
  • Zamboni P, Izzo M, Tognazzo S, et al. The overlapping of
    local iron overload and HFE mutation in venous leg ulcer pathogenesis.
    Free Radic Biol Med2006; 40:1869
  • Zamboni P, Tognazzo S, Izzo M, et al. Hemochromatosis
    C282Y gene mutation increases the risk of venous leg ulceration. J
    Vasc Surg
  • Wenk J, Foitzik A, Achterberg V, et al. Selective pick-up
    of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven
    induction of matrix degrading metalloproteinase 1 and lipid peroxidation in
    human dermal fibroblasts in vitro: a new dressing. Concept J Invest
  • Ackerman Z, Seidenbaum M, Loewenthal E, Rubinow A. Overload of iron
    in the skin of patients with varicose ulcers. Possible contributing role of
    iron accumulation in progression of the disease. Arch
    -8[Abstract/Free Full Text]
  • Zamboni P, Izzo M, Fogato L, Carandina S, Lanzara V. Urine
    haemosiderin: a novel marker to assess the severity of chronic venous disease.
    J Vasc Surg2003; 37:132
  • Coleridge Smith PD, Thomas P, Scurr JH, Dormandy JA. Causes of
    venous ulceration: a new hypothesis. BMJ1988; 296:1726
  • Takase S, Pascarella L, Lerond L, Bergan JJ, Schmid-Schonbein GW.
    Venous hypertension, inflammation and valve remodeling. Eur J Vasc
    Endovasc Surg
  • Wilkinson LS, Bunker C, Edwards JC, Scurr JH, Smith PD. Leukocytes:
    their role in the etiopathogenesis of skin damage in venous disease.
    J Vasc Surg1993; 17:669
  • Yeoh-Ellerton S, Stacey MC. Iron and 8-isoprostane levels in acute
    and chronic wounds. J Invest Dermatol2003; 121:918
  • Sullivan JL. Is stored iron safe? J Lab Clin
  • Herouy Y, Mellios P, Banderir E, et al. Inflammation in
    stasis dermatitis upregulates MMP-1, MMP-2 and MMP-13 expression. J
    Dermat Science
    2001; 25:198
  • Gurjar MV, Deleon J, Sharma RV, Bhalla RC. Role of reactive oxygen
    species in IL-1-stimulated sustained ERK activation and MMP-9 induction.
    Am J Phys2001; 281:H2568
  • Fog T. The topography of plaques in multiple sclerosis with special
    reference to cerebral plaques. Acta Neurol Scand1965; 15(Suppl):1
  • Putnam TJ. Lesions of `encephalomyelitis’and multiple sclerosis.
    Venous thrombosis as the primary alteration. JAMA1937; 108:1477[Abstract/Free Full Text]
  • Allen IV. The pathology of multiple sclerosis fact, fiction and
    hypothesis. Neuropathol Appl Neurobiol1981; 7:169[Medline]
  • Adams CW. Vascular aspects of multiple sclerosis. In: A
    Colour Atlas of Multiple Sclerosis and Other Myelin Disorders
    London. Wolfe Medical Publication, 1989:184
  • Adams CW, Poston RN, Buk SJ. Pathology, histochemistry and
    immunocytochemistry of lesions in acute multiple sclerosis. J
    Neurol Sci
  • Tan IL, van Schijndel RA, Pouwels PJ, et al. MR venography
    of multiple sclerosis. AJNR Am J Neuroradiol2000; 21:1039
    -42[Abstract/Free Full Text]
  • Horowitz AL, Kaplan RD, Grewe G, White RT, Salberg LM. The ovoid
    lesion: a new MR observation in patients with multiple sclerosis.
    AJNR Am J Neuroradiol1989; 10:303
  • Kidd D, Barkhof F, McConnell R, Algra PR, Allen IV, Revesz T.
    Cortical lesions in multiple sclerosis. Brain1999; 122:17
    -26[Abstract/Free Full Text]
  • Kermode AG, Thompson AJ, Tofts P, et al. Breakdown of the
    blood-brain barrier precedes symptoms and other MRI signs of new lesions in
    multiple sclerosis. Pathogenetic and clinical implications.
    Brain1990; 113:1477
    -89[Abstract/Free Full Text]
  • Kuenz B, Lutterotti A, Khalil M, et al. Plasma levels of
    soluble adhesion molecules sPECAM-1, sP-selectin and sE-selectin are
    associated with relapsing-remitting disease course of multiple sclerosis.
    J Neuroimmunol2005; 167:143
  • Martino G, Adorini L, Rieckmann P, et al. Inflammation in
    multiple sclerosis: the good, the bad, and the complex. Lancet
  • Haacke EM, Cheng NY, House MJ, et al. Imaging iron stores
    in the brain using magnetic resonance imaging. Magn Reson
  • Rubio JP, Bahlo M, Tubridy N, et al. Extended haplotype
    analysis in the HLA complex reveals an increased frequency of the HFE-C282Y
    mutation in individuals with multiple sclerosis. Hum
  • Fainardi E, Castellazzi M, Bellini T, et al. Cerebrospinal
    fluid and serum levels and intrathecal production of active matrix
    metalloproteinase-9 (MMP-9) as markers of disease activity in patients with
    multiple sclerosis. Mult Scler2006; 12:294
    -301[Abstract/Free Full Text]
  • Grant SM, Wiesinger JA, Beard JL, Cantorna MT. Iron-deficient mice
    fail to develop autoimmune encephalomyelitis. J Nutr2003; 133:2635
    -8[Abstract/Free Full Text]
  • Sfagos C, Makis AC, Chaidos A, et al. Serum ferritin,
    transferrin and soluble transferrin receptor levels in multiple sclerosis
    patients. Mult Scler2005; 11:272
    -5[Abstract/Free Full Text]

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