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62 Chapter 6/Inappropriate Leukocyte Activation in Venous Disease
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Interpretation of Data from Existing Studies
Endothelial adhesion is a normal physiological activity
of neutrophils and monocytes. During venous hypertension
the fall in blood fl ow to the lower limb and increase in
diameter of capillaries result in a fall in the shear rate in
cutaneous capillaries. This favors leukocyte adhesion, which
may be observed, even in control subjects, but is of greater
magnitude in patients with venous disease, presumably due
to the modifi cations that take place in the endothelium in
chronic venous disease.
It has been found that leukocyte-endothelial interaction
occurs during short-term venous hypertension (within 30
minutes) and that during this period neutrophil degranulation may be detected, releasing primary and secondary
granule enzymes into the region of the endothelium. At the
same time an increase in von Willebrand factor and soluble
endothelial adhesion molecules can be found in the leg
blood. These arguments apply to control subjects as well as
to patients, although the magnitude of change is always
FIGURE 6.7 From Reference 42. Increased procollagen type I-
expressing cells in lipodermatosclerotic (LDS) skin. The number of
positive dermal cells, as demonstrated by in-situ hybridization, was assessed
in skin sections from the control patients (CEAP class 0, n = 12), from
patients with chronic venous insuffi ciency but no clinical evidence of
LDS (CEAP class 2⁄3, n = 10), and from patients with LDS (CEAP class
4, n = 12). Data represent mean and SEM.
mediated by infl ammatory cell-derived factors but by additional profi brotic agents released in the skin as a consequence of chronic venous hypertension.
Some authors have studied the distribution of growth
substances and connective tissue proteins in skin biopsies
using immunohistochemical staining.43 In particular they
studied the pericapillary cuffs, which were once thought to
inhibit oxygen transfer to the tissues. The cuffs were positive
for actin, type IV collagen, factor XIIIa, and alpha 2macroglobulin, and there was increased TGF–β
1.
They
observed that TGF–β1 immunoreactivity was present within
the fi brin cuffs, but not in the provisional matrix in the ulcer
bed around the cuffs. These observations suggest that growth
factors critical in wound healing, such as TGF–β
, are
1
present within venous ulcers, but are abnormally distributed.
Their distribution within fi brin cuffs and colocalization
with extravasated plasma proteins, particularly alpha 2macroglobulin, which is a recognized scavenger molecule
for TGFβ and other growth factors, provides evidence for a
possible trapping of growth factors in venous ulcers. This
proposal has been advanced as a cause for failure of venous
leg ulcers to heal.
44
greater in the patients rather than the control subjects. The
research shows that when the venous system becomes
deranged, endothelial injury may be the result. Activated
leukocytes leave the lower limbs of control subjects following venous hypertension. In patients with venous disease,
these cells appear to remain in the lower limb, perhaps
attached to the abnormal endothelium.
The chronic changes seen in liposclerotic skin may be the
response to sustained, low-grade injury to the endothelium
by neutrophils and monocytes over many months or years.
The perivascular infi ltration of vessels in the papillary
dermis by macrophages and T-lymphocytes may simply be
a tissue response to the chronic infl ammatory processes
referred to earlier (see Figure 6.8). Endothelial activation is
seen during this phase with increased expression of endothelial adhesion molecules. This would favor the adhesion
of further leukocytes encouraging this process to continue.
The chronic infl ammatory process results in the release
of cytokines, which encourage vascular proliferation. VEGF
has been shown to be involved in this process. Whether this
is simply an associated phenomenon or crucial to subsequent
ulceration remains unclear at present. Extensive skin fi brosis, which is part of the clinical syndrome of lipodermatosclerosis, is a feature of chronic venous disease. The
macrophages present in the perivascular infl ammatory
process release TGFβ, and this in turn stimulates fi broblasts
to synthesize more collagen and connective tissue proteins.
The progression from the chronic skin damage to actual
ulceration remains diffi cult to understand. A possible explanation is that an initiating stimulus causes massive activation
of the peri-vascular macrophages, resulting in extensive
tissue and blood vessel destruction. This might occur spontaneously or minor trauma to the region may set in motion
the series of events that lead to ulcer formation.
Mechanisms of Ulceration 63
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Pericapillary
cuff
Capillary lumen
T-cell
VEGF
Vascular proliferation
FIGURE 6.8 Diagrammatic summary of fi ndings from many investigations in skin capillaries in patients with chronic
venous disease. The capillaries comprise endothelial cells showing activation. The vessels are surrounded by an infl ammatory cuff with a cellular infi ltrate, which includes macrophages. These and other cell types release a range of cytokines
that, among other things, produce vascular proliferation and skin fi brosis.
The data collected in the studies of neutrophil, monocyte,
and endothelial cell activity have so far failed to identify
major differences between those patients who develop skin
changes and are at risk of ulceration and those who do not.
Infl ammatory mechanisms are very complex and identifying
those which predispose to the development of skin changes
and ulceration will be a complex task.
Implications for Pharmacological
Treatment in Venous Disease
Although bandaging and stockings have been used effectively in the treatment of chronic venous insuffi ciency for
many years, modern pharmacological science may provide
assistance in healing venous ulcers and perhaps some insight
into the mechanisms of the disease.
Pentoxifylline has been used for the treatment of claudication for a number of years, with moderate success.
Its mechanism of action is probably through an effect on
inhibition of cytokine-mediated neutrophil activation.45 Its
effi cacy in healing venous leg ulcers has been reported in a
recent meta-analysis.46 Nine trials involving 572 adults were
included. Pentoxifylline plus compression is more effective
than placebo plus compression (relative risk of healing with
pentoxifylline 1.30, 95% confi dence interval 1.10–1.54).
Increased adhesion
molecule expression
Endothelial cell
Macrophage
TGF
β1
Fibrosis
MMPs
Fibroblast
This drug could be considered for use in patients with venous
leg ulceration when used in combination with compression.
Prostaglandin E
(PGE1) has a number of profound effects
1
on the microcirculation, including reduction of white cell
activation, platelet aggregation inhibition, small vessel vasodilatation, and reduction of vessel wall cholesterol levels.
Recently the results of a randomized, placebo-controlled,
single blind study in which 87 patients who had venous leg
ulcers has been reported.47 Patients were treated with compression bandaging and conventional wound management.
They also received treatment for 20 days with an infusion
of prostaglandin E1 analogue (Prostavasin, Schwarz Pharma)
or placebo. After four months, all ulcers were healed in the
active treatment group but only 32 of 38 in the placebo
group. This is a potentially useful drug but the limitation of
giving intravenous infusions restricts it applicability.
Laurent et al. investigated micronized purifi ed fl avonoid
fraction (MPFF)48 and showed that this drug reduced the
symptoms of venous disease (aching, itching, feeling of
swelling) and also reduced ankle edema. More recently
MPFF has been studied for its effects on venous leg ulcer
healing. A meta-analysis has been published in which fi ve
prospective, randomized, controlled studies involving 723
patients with venous ulcers were included.49 Patients were
treated with compression bandaging and local wound care
in all cases. In two studies MPFF was compared to placebo
64 Chapter 6/Inappropriate Leukocyte Activation in Venous Disease
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and in three studies MPFF was compared to standard treatment alone. At six months, the chance of healing ulcer was
32% better in patients treated with adjunctive MPFF than in
those managed by conventional therapy alone. The main
benefi t of MPFF was present in the subgroup of ulcers
between 5 and 10 cm2 in area and those present for six to 12
months duration. MPFF therefore may be a useful drug to
combine with compression management in countries where
it is licensed.
CONCLUSIONS
The precise mechanisms through which venous hypertension causes ulceration remain to be discovered. There is
clear evidence of leukocyte activation in venous disease and
many infl ammatory mechanisms are upregulated in the skin.
So far it has been impossible to say which of these is the
main cause of the problem and which are simply a response
to the infl ammatory process. Drugs that mitigate leukocyte
activation appear to benefi t ulcer healing. A better understanding of the initiating processes may lead to improvements in the management of patients with venous
ulceration.
References
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Prevalence of leg ulceration in a London population, QJM. 2004. 97:
431–437.
2. Evans CJ, Fowkes FG, Ruckley CV, Lee AJ. Prevalence of varicose
veins and chronic venous insuffi ciency in men and women in the
general population: Edinburgh Vein Study, J Epidemiol Community
Health. 1999. 53: 149–153.
3. Callam MJ, Ruckley CV, Harper DR, Dale JJ. Chronic ulceration of
the leg: Extent of the problem and provision of care, BMJ. 1985. 290:
1855–1856.
4. Baker SR, Stacey MC, Singh G, Hoskin SE et al. Aetiology of chronic
leg ulcers, Eur J Vasc Surg. 1992. 6: 245–251.
5. Nelzén O, Bergqvist D, Lindhagen A, Hallböök T. Chronic leg ulcers:
An underestimated problem in primary health care among elderly
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6. Nicolaides AN, Zukowski A, Lewis R, Kyprianou P, Malouf GM.
Venous pressure measurements in venous problems. In: Surgery of the
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7. Dodd H, Cockett FB. The pathology and surgery of the veins of the
lower limb. Edinburgh: Churchill Livingstone. 1976.
8. Hoare MC, Nicolaides AN, Miles CR, Shull K, Jury RP, Needham T,
Dudley HAF. The role of primary varicose veins in venous ulceration,
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9. Browse NL, Burnand KG. The cause of venous ulceration, Lancet.
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Three tests of microcirculatory function in the evaluation of treatment
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12. Thomas PRS, Nash GB, Dormandy JA. White cell accumulation in the
dependent legs of patients with venous hypertension: A possible mechanism for trophic changes in the skin, Br Med J. 1988. 296: 1693–
1695.
13. Braide M, Amundson B, Chien S, Bagge U. Quantitative studies of
leucocytes on the vascular resistance in a skeletal muscle preparation,
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14. Engler RL, Dahlgren MD, Peterson MA, Dobbs A, SchmidSchoenbein GW. Accumulation of polymorphonuclear leucocytes
during three hour myocardial ischemia, Am J Physiol. 1986. 251:
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15. Romson JL, Hook BG, Kunkel SL, Abrams GD, Schork MA, Lucchesi
BR. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog, Circulation. 1983. 67: 1016–1023.
16. Wilson JW. Leucocyte sequestration and morphologic augmentation
in the pulmonary network following haemorrhagic shock and related
forms of stress, Adv Microcirc. 1972. 4: 197–232.
17. Linas SL, Shanley PF, Whittenburg D, Berger E, Repine JE. Neutrophils accentuate ischemia-reperfusion injury in isolated perfused rat
kidneys, Am J Physiol. 1988. 255: F728–F735.
18. Yamakawa T, Suguyama I, Niimi H. Behaviour of white blood cells
in microcirculation of the cat brain cortex during hemorrhagic shock.
Intravital microscopic study. Int J Microcirc: Clin Exp. 1984. 3: 554.
19. Braide M, Blixt A, Bagge U. Leukocyte effects on the vascular resistance and glomerular fi ltration of the isolated rat kidney at normal and
low fl ow rates, Circulatory Shock. 1986. 20: 71–80.
20. Weissman G, Smolen JE, Korchak HM. Release of infl ammatory
mediators from stimulated neutrophils, N Engl J Med. 1980. 303:
27–34.
21. Babior BM. Oxidants from phagocytes: Agents of defense and destruction, Blood. 1984. 64: 959–966.
22. Coleridge Smith PD, Thomas P, Scurr JH, Dormandy JA. Causes of
venous ulceration: A new hypothesis, Br Med J. 1988. 296: 1726–1727.
23. Shields DA, Andaz S, Abeysinghe RD, Porter JB, Scurr JH, Coleridge
Smith PD. Neutrophil activation in experimental ambulatory venous
hypertension, Phlebology. 1994. 9: 119–124.
24. Shields D, Andaz SK, Timothy-Antoine CA, Scurr JH, Porter JB.
CD11b/CD18 as a marker of neutrophil adhesion in experimental ambulatory venous hypertension. In: Phlebology ’95, D Negus, G Jantet, PD
Coleridge Smith, eds. Phlebology. 1995. Suppl. 1: 108–109.
25. Saharay M, Shields DA, Porter JB, Scurr JH, Coleridge Smith PD.
Leukocyte activity in the microcirculation of the leg in patients with
chronic venous disease, J Vasc Surg. 1997. 26: 265–273.
26. Shields DA, Andaz S, Sarin S, Scurr JH, Coleridge Smith PD. Neutrophil activation in experimental venous hypertension, Phlebologie.
1993. 46: 687–689.
27. Shields DA, Andaz S, Abeysinghe RD, Porter JB, Scurr JH, Coleridge
Smith PD. Plasma lactoferrin as a marker of white cell degranulation
in venous disease, Phlebology. 1994. 9: 55–58.
28. Shields DA, Andaz SK, Sarin S, Scurr JH, Coleridge Smith PD. Plasma
elastase in venous disease, Br J Surg. 1994. 81: 1496–1499.
29. Shields D, Saharay M, Timothy-Antoine CA, Porter JB, Scurr JH.
Neutrophil CD11b expression in patients with venous disease. In: Phlebology ’95, Negus D, Jantet G, Coleridge Smith PD, eds. Phlebology.
1995. Suppl. 1: 108–109.
30. Scott HJ, McMullin GM, Coleridge Smith PD, Scurr JH. A histological study into white blood cells and their association with lipodermatosclerosis and ulceration, Br J Surg. 1990. 78: 210–211.
31. Wilkinson LS, Bunker C, Edwards JC, Scurr JH, Coleridge Smith PD.
Leukocytes: Their role in the etiopathogenesis of skin damage in
venous disease, J Vasc Surg. 1993. 17: 669–675.
32. Burnand KG, Whimster I, Naidoo A, Browse NL. Pericapillary fi brin
in the ulcer-bearing skin of the leg: The cause of lipodermatosclerosis
and venous ulceration, Br Med J. 1982. 285: 1071–1072.
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33. Haselbach P, Vollenweider U, Moneta G, Bollinger A. Microangiopathy in severe chronic venous insuffi ciency evaluated by fl uorescence
video-microscopy, Phlebology. 1986. 1: 159–169.
34. Howlader MH, Coleridge Smith PD. Microangiopathy in chronic
venous insuffi ciency: Quantitative assessment by capillary microscopy, Eur J Vasc Endovasc Surg. 2003. 26: 325–331.
35. Veraart JC, Verhaegh ME, Neumann HA, Hulsmans RF, Arends JW.
Adhesion molecule expression in venous leg ulcers, Vasa. 1993. 22:
213–218.
36. Herrick SE, Sloan P, McGurk M, Freak L, McCollum CN, Ferguson
MW. Sequential changes in histologic pattern and extracellular matrix
deposition during the healing of chronic venous ulcers, Am J Pathol.
1992. 141: 1085–1095.
37. Scott HJ, McMullin GM, Coleridge Smith PD, Scurr JH. A histological study into white blood cells and their association with lipodermatosclerosis and ulceration, Br J Surg. 1990. 78: 210–211.
38. Falanga V, Kruskal J, Franks JJ. Fibrin- and fi brinogen-related antigens
in patients with venous disease and venous ulceration, Arch Dermatol.
1991. 127: 75–78.
39. Saharay M, Shields DA, Georgiannos SN, Porter JB, Scurr JH,
Coleridge Smith PD. Endothelial activation in patients with chronic
venous disease, Eur J Vasc Endovasc Surg. 1998. 15: 342–349.
40. Pardoe HD. The expression of angiogenic growth factors in the skin
of patients with chronic venous disease of the lower limb. MSc Thesis,
University College London. September 1996. pp. 1–61.
41. Pappas PJ, You R, Rameshwar P, Gorti R, DeFouw DO, Phillips CK
et al. Dermal tissue fi brosis in patients with chronic venous insuffi -
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gene expression and protein production, J Vasc Surg. 1999. 30: 1129–
1145.
42. Degiorgio-Miller AM, Treharne LJ, McAnulty RJ, Coleridge Smith
PD, Laurent GJ, Herrick SE. Procollagen type I gene expression and
cell proliferation are increased lipodermatosclerosis, Br J Dermatol.
2005. 152: 242–249.
43. Higley HR, Ksander GA, Gerhardt CO, Falanga V. Extravasation of
macromolecules and possible trapping of transforming growth factorbeta in venous ulceration, Br J Dermatol. 1995. 132: 79–85.
44. Falanga V, Eaglstein WH. The “trap” hypothesis of venous ulceration,
Lancet. 1993. 341: 1006–1008.
45. Sullivan GW, Carper HT, Novick WJ, Mandell GL. Inhibition of the
infl ammatory action of interleukin-1 and tumour necrosis factor (alpha)
on neutrophil function by pentoxifylline, Infect. Immunol. 1988. 56:
1722–1729.
46. Jull AB, Waters J, Arroll B. Pentoxifylline for treating venous leg
ulcers, Cochrane Database Syst Rev. 2002. (1): CD001733.
47. Milio G, Mina C, Cospite V, Almasio PL, Novo S. Effi cacy of the
treatment with prostaglandin E-1 in venous ulcers of the lower limbs,
J Vasc Surg. 2005. 42: 304–308.
48. Laurent R, Gilly R, Frileux C. Clinical evaluation of a venotropic drug
in man. Example of Dafl on 500 mg. Int Angiol. 1988. 7(2 Suppl):
39–43.
49. Coleridge-Smith P, Lok C, Ramelet AA. Venous leg ulcer: A metaanalysis of adjunctive therapy with micronized purifi ed fl avonoid fraction, Eur J Vasc Endovasc Surg. 2005. 30: 198–208.
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CHAPTER
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7
Molecular Basis of Venous Insuffi ciency
GEERT W. SCHMID-SCHÖNBEIN
INTRODUCTION
Chronic venous disease (CVD) of the lower limb reaches
its most severe form as varicose veins and venous leg ulcers.
But before reaching such endstages, CVD goes through a
range of manifestations that also include edema and skin
changes (venous eczema, ankle skin hyperpigmentation,
atrophie blanche, and lipodermatosclerosis). Robust evidence has now been gathered to indicate that CVI is an
infl ammatory disease. This recognition has facilitated understanding the pathophysiological processes that underlie
these diverse manifestations, particularly at the cellular and
molecular levels. The recognition that an infl ammatory
process may be involved has opened a range of opportunities
for development of novel interventions. But it also has
opened the door to an analysis that may lead us toward
the trigger mechanisms for infl ammation in CVD; That is,
an understanding of the origin of the earliest manifestations (like telangiectasia) and the key to future design of
preventions.
TISSUE RESTRUCTURING
The infl ammatory process and lesions in different stages
of CVD involve the superfi cial layers of the skin but possibly also various venous valves, some of which may be
located deep. Capillary density may vary in liposclerotic
skin. For example, some areas of white atrophy (atrophie
blanche) may show a complete loss of capillaries in some
areas whereas in others, the capillaries appear dilated, elongated, coiled, and tortuous.
laries have been observed. These include irregular wavy
shapes of the luminal surface, intracellular edema, and
1
Endothelial lesions in the capil-
increased intra-cytoplasmic vesicles. Basement membranes
appear to be completely fused with the surrounding tissue.
Pericapillary spaces are fi lled with a fl uid that is rich in
cellular fragments and proteins. Pericytes and fi brin cuffs
have been observed in the pericapillary spaces and around
the capillaries.2 In capillaries per se there is red cell packing,
white blood cells, and platelet aggregates that may fi ll the
capillary lumen and may be found in the pericapillary
tissue.
In addition there is failure of valves through dilation of
the venous wall and remodeling of the valve leafl ets dilation
of the valvular annulus, bulging and stretching of valve
leafl ets, commissural dilation, shortening, tearing and perforation of leafl ets, and, fi nally, complete destruction of the
3,4
valve.
chemical studies of valves and the venous wall have revealed
leukocytes adhering and transmigrating into the venous
wall.
Furthermore, ultrastructural and immunohisto-
5,6
EARLY MANIFESTATIONS
OF INFLAMMATION
The suggestion that infl ammation may be involved in
CVD comes from the evidence for elevated endothelial permeability,2 a process that tends to involve inter-endothelial
junctions. This can readily be observed in acute models of
venous hypertension (see Figure 7.1). Although there are a
large number of mediators (histamine, platelet activating
factor, cytokines) that have the ability to elevate endothelial
permeability, most of them when acutely applied to a tissue
act transiently via mechanisms that involve nitric oxide,
actin polymerization, and selected small GTPases.
serves as an indication that already an early event in CVD
8–15
7
This
The Vein Book
67
All rights of reproduction in any form reserved.
Copyright © 2006, Elsevier Inc.
68 Chapter 7/Molecular Basis of Venous Insuffi ciency
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Macromolecular Leakage
(Monastal Blue B)
50 µm
Microhemorrhage
FIGURE 7.1 Short-term venous pressure elevations cause elevated
microvascular permeability and red cell diathesis. Intravital micrographs
showing evidence for elevation of venular permeability (detected by monastral blue escape across the endothelium, top panel) and extravasation of
red cells across postcapillary venules in rat mesentery (bottom panel) after
occlusion of a larger downstream venule (about 500 μm) for 1 hour fol-
lowed by 1 hour reperfusion. Adapted from (63).
is possibly driven by mechanisms that have the ability to
produce a chronic infl ammatory state. The reduction of
endothelial permeability may be a target of opportunity for
early intervention since it may interrupt the infl ammatory
cascade at one of its earlier steps. Elevation of endothelial
permeability with opening of leakage sites between endothelial cells depends on the inter-endothelial adhesion molecule VE-cadherin,
on the endothelial cells,17 on the specifi c venular genotypes,18
and on exercise training.
The link between infl ammation and skin changes may be
by way of the proteolytic enzymes, including Ca/Zndependent endoproteinases (matrix metalloproteinases,
16
an enzyme sensitive glycocalyx layer
19
TISSUE REMODELING AND
ENZYMATIC ACTIVITY
MMPs) and serine proteinases. Chronic dermal ulcers are
characterized by excessive proteolytic activity, which
degrades extracellular matrix and growth factors and their
receptors. The MMP family of proteases (all of them can be
inhibited by metal chelation) are positioned on extracellular
matrix proteins in an inactive proform. They play an im-
20
portant role in cell differentiation and development.
MMPs
may be released from preexisting pools (cytoplasmic
granules) upon stimulation and endocytosis or may be newly
synthesized by several types of cardiovascular cells.21 The
inactive pro-enzymes can be activated by other proteinases,
including those produced by mast cells.22 MMPs have multiple binding sites but cleave collagen at unique sites.23 The
MMP levels in wound fl uids from chronic wounds tend to
be signifi cantly higher than from acute wounds and healing
is associated with reduced MMP activity.
24
The expression levels of MMPs can be controlled by
mechanical stretch (strain) (see Figure 7.2), but in a way that
depends on the time course of the strain. This has been
studied on smooth muscle cells in an in-vitro chamber
subject to oscillatory and constant strain. Stationary strain
signifi cantly increases MMP-2 mRNA levels at all time
points, whereas cyclic strain decreases it after 48 h. Both
secreted and cell-associated pro-MMP-2 levels are increased
by stationary strain at all times, whereas cyclic strain
decreases secreted levels after 48 hours. MMP-9 mRNA
levels and pro-MMP-9 protein are increased after 48 hours
of stationary strain compared with both no strain and cyclic
25
strain.
Neutrophils, besides a rich protease population in their
primary and secondary granules, also have several MMPs.
There is gelatinolytic activity discharged by MMP-9, MMP8 a neutrophil collagenase, leukolysin a membrane-type
MMP.26 Evidence from knock-out experiments suggests that
MMP-9 acts upstream of neutrophil elastase by proteolytically inactivating neutrophil elastase inhibitor a1-PI27 and
that it can activate other MMPs.28 Extracellular MMP inducer
(EMMPRIN; CD147) has been observed to increase MMP
expression, and membrane type 1 MMP (MT1-MMP) has
been implicated in the activation of MMPs. Venous leg
ulcers have elevated expression of EMMPRIN, MMP-2,
MT1-MMP, and MT2-MMP.
29
Clearly, these proteases will remain of major interest
because of their involvement in both the infl ammatory
reaction and the remodeling of cutaneous tissue.30 Current
interest is focused on MMP1, MMP2, MMP9, MMP12,
MMP13, TIMP1, and TIMP2.
31–33
The overexpression of
MMP-3 (stromelysin-1) and MMP-13 (collagenase-3) is
associated with nonhealing wounds.34 Recent studies showed
an increased expression of MMP2 and TIMP1 in liposcler-
30,35
otic skin,
in venous leg ulcers,29 and in wound fl uid
from nonhealing venous ulcers.37 Expression of MMP9 has
been observed to be upregulated on the edges of intractable
venous ulcers35 and the rate of MMP-9 activation in plasma
Tissue Remodeling and Enzymatic Activity 69
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A
B
FIGURE 7.2 MMP activation in the early phases of venous hypertension. (A) Immunohistochemical labeling of
paraffi n section of rat femoral vein after placement of an arteriolar/venular fi stula for one week to raise venous pressure.
The vein still has an intact valve but the distension leads to extensive expression of MMP2 (detected by deposition of
biotin avidin enzymatic reaction product (dark brown color) in the media of the vein. (B) Gel electrophoresis zymography of saphenous vein segments subjected to the hypertension by placement of an arterial/venous fi stula. The four
left lanes are from 1 day and 7 day; each experimental group is paired with the control represented by the contralateral
vein; the center “H” lane represents human MMP-2 and MMP-9 standards; the four right lanes are from the later time
points at 21 and 42 days; each experimental group is paired with the control represented by the contralateral vein. Vein
segments subjected to the fi stula present signifi cantly higher level of MMP-9 at 7 days from the creation of the fi stula.
Higher levels of pro-MMP2 and MMP-2 were observed at 21 and 42 days from the creation of the fi stula. Adapted from
(148).
of patients with severe CVD is elevated.36 Levels of TIMP2 are lower in lipodermatosclerotic skin and ulcers.
30,37
Unrestrained MMP activity may contribute to extracellular matrix protein breakdown that impairs healing. Proteolytic reactions may be even of greater importance than
other cytotoxic reactions, such as oxygen free radical
production.
The skin hyperpigmentation seen in lipodermatosclerosis
may not just be an innocent byproduct of capillary hyperpermeability. The extravasation of red blood cells leads to
oxidative stress38 as well as elevated ferritin and ferric iron
levels in affected skin that causes hydroxyl radical forma-
39–41
tion,
possible MMP activation, and development of a
local microenvironment that exacerbates tissue damage
and delays healing.
42
Consistent with this, the hemochroma-
tosis C282Y mutation (a common genetic defect of iron
metabolism) is associated with a near 7-fold increase in risk
of ulceration among CVD patients.
43
A critical issue are not only the mechanisms that cause
expression of these proteases, but also the mechanisms that
cause their activation and the often lack of anti-protease
activity. Among the mechanisms that have been proposed to
activate MMPs is plasmin,44 serine proteaseses like trypsin,45
MMP-3, and MMP-13.33 These may involve indirect pathways, indicating the possible complexity of the activation
process involved. Plasmin stimulates pro-MMP enzyme
conversion to the active form. Plasmin hyperactivity due to
decreased plasminogen activator inhibitor-1 (PAI-1) may
44
thus cause MMP overactivity.
But these are important
regulatory pathways that need to be clarifi ed to understand
the activation of the proteolytic process that causes restructuring in venous disease (see later).
70 Chapter 7/Molecular Basis of Venous Insuffi ciency
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LEUKOCYTE ENTRAPMENT
AND MOLECULAR
ADHESION MECHANISMS
The modern developments of the pathophysiological
basis of the skin changes in CVD can perhaps be traced back
to the simple observation that leukocytes have quite different biomechanical properties than the red cells. Even though
they are clearly in the minority, they contribute to many
microvascular events.
in the microcirculation and thus it is highly signifi cant that
blood returning from feet that have been passively dependent for 40 to 60 minutes is relatively depleted of leukocytes, especially in patients with CVD.
easily trapped in the microcirculation due to their stiff cytoplasmic properties and their ability to express membrane
adhesion molecules, further enhanced by the fact that these
properties are changed dramatically after activation.51 This
subtle mechanism suggests that leukocytes accumulate in
the lower extremity under conditions of high venous pressure. It is possible that the accumulation in microvessels is
due to the fact that the cells are already activated in the
central circulation,
lated in the circulation by a mechanism that could be located
outside of the venous network of the lower extremities.
Alternatively leukocytes become entrapped in the skin
microcirculation by adhesion to the endothelium, which is
activated by a local process not necessarily dependent on the
same mechanisms that causes leukocyte activation. The leukocyte adhesion is to the membrane of the endothelium of
small vessels, especially capillaries and post-capillary
venules. The accumulated leukocytes became activated
and led to the suggestion that an infl ammatory reaction is
important in provoking skin changes in CVD. Numerous
recent studies have added weight to that suggestion.
Most interesting in this respect are the observations by
Coleridge-Smith and his colleagues, showing degranulation
of the leukocytes with an increase of neutrophil elastase and
lactoferrin, markers of neutrophil activation, in patients
under transient conditions of venous hypertension and with
chronic venous insuffi ciency.
effective activators of other proenzymes, such as MMPs.
The circulating mononuclear cells of patients with CVD also
have reduced degree of proliferation in response to a mitogenic challenge (staphylococcal enterotoxins) and thus a
reduced capacity for wound healing.
Skin biopsies from CVD limbs show elevated numbers
of macrophages, T-lymphocytes, and mast cells.
is the same pattern as observed in both acute
chronic64 experimental rat models of venous hypertension, with elevated levels of tissue leukocytes in skin
samples from affected limbs, but not from sham-operated
controls.
46,47
One of them is local accumulation
48–50
Leukocytes are
6,52
which means that they may be stimu-
56–58
These enzymes may be
59
60,61
62,63
53–55
This
and
The molecular mechanisms involved in leukocyte adhesion and activation in CVD patients are now beginning to
be elucidated. For example, transient binding of L-selectin
on the leucocyte surface to E-selectin on endothelial cells is
involved in leukocyte “rolling” along the endothelial surface.
However, when leukocytes are activated they shed Lselectin into the plasma and express molecules of the
integrin family, including CD11b that binds to intercellular
adhesion molecule-1 (ICAM-1). Integrin binding promotes
fi rm adhesion of leukocytes, the starting point for their
migration out of the vasculature and degranulation.
The evidence in CVD suggests that a variety of membrane adhesion molecules on endothelial cells and leukocytes (ICAM-1, vascular cell adhesion molecule-1, LFA-1,
VLA-4, Mac 1, and others) appear to facilitate the adhesion
and stimulate projection of pseudopodia as a requirement for
transmigration of the leukocyte into the venous wall.
leukocyte infi ltration of the venous parenchyma is accompanied by remodeling of the extracellular matrix, a process
that is ultimately responsible for the destruction of the
venous valves.
At the same time, plasma levels of soluble L-selectin
increase, refl ecting the shedding of these molecules from
leukocyte membranes during leukocyte-endothelial adhesion.69 Similarly, basal plasma levels of the adhesion molecules ICAM-1, endothelial leukocyte adhesion molecule-1
(ELAM-1), and vascular cell adhesion molecule-1 (VCAM-
1) are higher in CVD patients than controls and increased
signifi cantly in response to venous hypertension provoked
by standing.
67
In addition to local factors operating in relation to venous
hypertension, CVD patients have a tendency for systemically elevated leukocyte adhesion. Plasma obtained from
CVD patients induced higher degrees of activation (assessed
by oxygen-free radical production and pseudopod formation) in healthy, naive granulocytes than did plasma taken
from normal subjects.6 The nature of the plasma factor
responsible is presently unknown.
CYTOKINES
Even though it is well recognized that cytokines are
part of the infl ammatory reaction in CVD
picture exists about their exact role. For example treatment
with granulocyte/monocyte colony stimulating factor
(GM-CSF) leads to mixed results in ulcer healing. TNFα,
whose expression is enhanced in many infl ammatory
reactions, stimulates the expression of infl ammatory adhesion molecules, the synthesis and release of other cytokines,
and the chemotaxis of neutrophils and macrophages. The
expression of TNFα appears to be upregulated in patients
65,70,71
65–68
The
no clear
Trigger Mechanisms for Cell Activation 71
https://t.me/med1917
with venous ulcers and healing of the ulcer may reduce the
level of TNFα.
72
GROWTH FACTORS
In lipodermatosclerosis, the skin capillaries become
elongated and tortuous,73 and may take on a glomerular
appearance in more advanced skin changes,74 with proliferation of the capillary endothelium. Vascular endothelial
growth factor (VEGF) is an obvious candidate for involvement in these changes, and has been shown to increase
microvascular permeability.75 Plasma levels of VEGF
increase during the venous hypertension-induced short-term
standing in both normal subjects and CVD patients, but are
higher in patients than in normal controls.76 Furthermore,
plasma VEGF levels are higher in CVD patients with skin
changes than in CVD patients with normal skin.77 VEGF
also is known to induce the expression of adhesion molecules, such as ICAM-1, VCAM-1, and E-Selectin.78 Both
VEGF expression and its receptor expression (Flk-1/KDR)
are upregulated under variations of blood shear stress and
with the infl ammatory reaction.
increases with the severity of the disease and the CEAP
score.
Another feature of the skin changes associated with CVD
is dermal tissue fi brosis. Transforming growth factor-β1
(TGF-β1) is a known fi brogenic cytokine. Immunocytochemical analysis of punch biopsy specimens showed that
skin from the lower calf of CVD patients had signifi cantly
elevated active TGF-β1 levels compared with normal skin
or skin taken from the thigh region of the same patients.
The TGF-β1 was located in leukocytes, fi broblasts, and on
collagen fi brils, and Pappas proposed that activated leukocytes migrate out of the vasculature and release TGF-β1,
stimulating increased collagen production by dermal fi broblasts and leading to dermal fi brosis.
It is evident that analysis of the infl ammatory cascade in
venous disease has just begun. Many details will be revealed
in the future especially at the molecular level, which will
open new opportunities for intervention. But I like to draw
attention to another issue important in the contact of infl ammation. The infl ammatory cascade serves under normal conditions as a repair mechanism after tissue injury. One should
ask then, why are there skin lesions, which for long periods
of time do not proceed to a resolution of the infl ammation?
There may be mechanisms that serve to maintain an infl ammatory state, but it is possible that the trigger mechanism
that set into motion the fi rst step in the infl ammatory cascade
is never quite eliminated in chronic conditions. Thus, we
need to engage in an analysis of possible trigger mechanisms
for infl ammation in CVD.
79,80
The level of VEGF
81
61,81
TRIGGER MECHANISMS
FOR CELL ACTIVATION
There are a large number of primary mechanisms that
may serve to activate cells and stimulate an infl ammatory
cascade in the circulation. It is helpful to classify these
mechanisms into several general categories:
a) Positive feedback mechanisms: There exists a class of
infl ammatory reactions that are mediated by direct
action of plasma infl ammatory stimulators (oxygen free
radicals,82 platelet activating factor (PAF),83 cytokines
(e.g., TNF-α, IL-1, IL-8),84 complement fragments,85
endotoxins, coagulation and fi brinolytic factors,
leukotrienes, thrombin, and oxidized LDL). The list of
infl ammatory mediators is long, and may in part be
triggered by trauma or by bacterial, viral, or fungal
sources.
b) Negative feedback mechanisms: An alternative pathway
for cell upregulation in the microcirculation is by
depletion of anti-infl ammatory factors. This list is
somewhat shorter and includes nitric oxide, adenosine,
glucocorticoids, selected cytokines (e.g., IL-10),
estrogen,87 and some proteins like albumin.
c) Contact activation: A specialized form of cell
activation by membrane contact has been proposed in
the form of juxtacrine activation.88 A nonactivated
endothelial cell may be stimulated during membrane
contact by an activated leukocyte and vice versa, for
example, by oxygen free radical production in the
membrane contact region between the cells and by
formation of platelet activating factor (PAF) and
other bioactive lipids.
d) Activation by mechanotransduction: These mechanisms
for cell activation involve fl uid shear stress (force per
unit area parallel to a surface) and normal stress (for
per unit area normal to a surface, i.e., pressure). These
two mechanical stresses likely play an important role in
venous disease (see later).
e) Activation by physical transients: Transients of gas
concentrations (like oxygen, carbon dioxide, etc.) or
also temperature transients have the ability to stimulate
cell activation irrespective of the direction of the
transient (up or down) but dependent on the magnitude
of the transient.
89–93
f) Activation by hormonal pathways: Candidates are
progesterone, insulin, and others
94–98
but their action
depends on cofactors.
g) Genetic mechanisms: There may be a large number,
some associated with single nucleotide perturbations
(SNPs), defects in transcription, and even lack of gene
expression, expression of spice variants, to protein
folding.
86
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