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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_3829_Библиотеки_им_академика_М_И_Перельмана
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and 62% for females if one parent is a ected, and 20% when
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neither parent is a ected. ese data suggest an autosomal
dominant with variable penetrance mode of genetic transmission. e decreased incidence in males with an a ected
parent and the spontaneous development in patients without a ected parents suggests that males are more resistant
to varix formation and that other multifactorial etiologies
in patients with predispositions to the disease must exist.
To further elucidate the genetic component of the disease,
molecular analyses with gene chip technologies is required.
e chromosome responsible for the disease and its protein
byproducts are currently unknown.
An injury to the venous endothelium or local procoagulant environmental factors leads to thrombus formation
in the venous system. It is currently well accepted that a
venous thrombus initiates a cascade of in ammatory events
10
that contributes to or causes vein wall brosis.
rombus
formation at venous con uences and valve pockets leads to
activation of neutrophils and platelets. Activation of these
cells leads to formation of in ammatory cytokines, procoagulants, and chemokines leading to thrombin activation
and further clot formation. Production of in ammatory
mediators creates a cytokine/chemokine gradient leading
to leukocyte invasion of the vein wall at the thrombus wall
interface and from the surrounding adventitia. Upregulation
of adhesion molecules perpetuates this process, eventually
leading to vein wall brosis, valvular destruction, and altera-
10,11
tion of vein wall architecture.
Although the mechanisms
associated with vein wall damage secondary to venous
thrombosis are beginning to be unraveled, the majority of
varicose veins occur in patients with no prior history of deep
venous thrombosis. e etiology of primary varicose veins
continues to be a mystery.
VEIN WALL ANATOMY,
HISTOPATHOLOGY, AND
FUNCTIONAL ALTERATIONS
Figure8.1 Electron micrograph of normal GSV (Mag 11,830×).
Note organized structure of alternating smooth muscle cells (long
arrows) with spindle-shaped contractile phenotype, interspersed by
longitudinally arranged collagen bundles (short arrows).
rather than spindle-shaped, and demonstrate numerous
collagen-containing vacuoles imparting a secretory pheno-
15
type (see Figure8.2).
What causes SMCs to dedi erentiate from a contractile to a secretory phenotype is currently
unknown. Ascher etal. theorized that SMC dedi erentia-
18,19
tion may be related to dysregulation of apoptosis.
ese
investigators reported a decrease in the proapoptotic mediators Bax and PARP (poly ADP-ribose polymerase) in the
adventitia of varicose veins compared with normal veins.
Although no di erence in these mediators was observed in
the media or intima of varicose veins, a decrease in SMC
turnover was postulated as a possible cause for the increase
in secretory phenotype. Increased phosphorylation of the
retinoblastoma protein, an intracellular regulator of cellular
proliferation and di erentiation, has been observed in vari-
13
cose veins, and may similarly contribute to this process.
Vein wall remodeling has been observed consistently
12,14-17,20
in histologic varicose vein specimens.
Gandhi et al.
Whatever the initiating event, several unique anatomic and
biochemical abnormalities have been observed in patients
with varicose veins. Normal and varicose GSVs are characterized by three distinct muscle layers within their walls.
e media contains an inner longitudinal and an outer circular layer, and the adventitia contains a loosely organized
outer longitudinal layer.
layers are composed of smooth muscle cells (SMCs), which
appear spindle-shaped (contractile phenotype) when examined with electron microscopy (see Figure8.1).
lie in close proximity to each other, are in parallel arrays,
and are surrounded by bundles of regularly arranged collagen bers. In varicose veins, the orderly appearance of the
muscle layers of the media is replaced by an intense and
disorganized deposition of collagen.
its separate the normally closely opposed SMCs and are
particularly striking in the media. SMCs appear elliptical
12–14
In normal GSVs, these muscle
15
ese cells
15–17
Collagen depos-
68 • BASIC CONSIDERATIONS
Figure8.2 Electron micrograph of varicosed GSV (Mag 4240×). Smooth
muscle cells exhibit prominent vacuoles (arrows) and an elliptical
appearance consistent with a secretory phenotype. Smooth muscle cells
are separated by di usely deposited collagen bundles, which impart a
disorganized architectural appearance to the veinwall.
quantitatively demonstrated an increase in collagen content
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and a decrease in elastin content compared with normal
20
GSVs.
e net increase in the collagen/elastin ratio suggested an imbalance in connective tissue matrix regulation.
As a result, several investigators have observed alterations in
matrix metalloproteinase and brinolytic activity in varicose
veins. TIMP-1 and MMP-1 protein levels are increased at
the saphenofemoral junction compared with normal con-
21
trols, whereas MMP-2 levels are decreased.
No overall
di erences in MMP-9 protein or activity levels have been
identi ed, however, the number of cells expressing MMP-9
by immunohistochemistry has been reported to be elevated
22,23
in varicose veins compared with normal veins.
ere are
con icting reports regarding the role of plasmin activators
and their inhibitors. Shireman etal. reported that uPA (urokinase plasminogen activator) levels are increased three to ve
times compared with normal controls in the media of vein
24
specimens cultured in an organ bath system.
No di erences
were noted in tPA (tissue plasminogen activator) or PAI-1
(plasmin activator inhibitor-1) levels. However, other investigations have reported a decrease in uPA and tPA activity by
22,25
enzyme zymography in varicose veins.
ese data suggest
that the plasminogen activators may play a role in matrix
metalloproteinase activation leading to vein wall brosis and
varix formation; however, further research into the mechanisms regulating vein wall brosis clearly are needed.
What e ect vein wall brosis has on venous function
needs further elucidation. e contractile responses of
varicose and normal GSV rings to noradrenaline, potassium chloride, endothelin, calcium ionophore A23187,
angiotensin II, and nitric oxide have been evaluated by
26,27
several investigators.
ese studies have demonstrated
decreased contractility of varicose veins when stimulated
by noradrenaline, endothelin, and potassium chloride.
Similarly, endothelium-dependent and - independent relaxations a er A23187 or nitric oxide administration were
diminished compared with normal GSVs, respectively.
e mechanisms responsible for decreased varicose vein
27,28
contractility appear to be receptor mediated.
Utilizing
Sarafotoxin S6c (selective pharmacologic inhibitor of endothelin B) and competitive inhibition receptor assays with
131
I-endothelin-1, a decrease in endothelin B receptors
have been observed in varicose veins compared with nor-
28
mal GSVs.
Feedback inhibition of receptor production
secondary to increased endothelin-1 is postulated to mediate the decreased receptor content in varicose vein walls.
Other possible mechanisms for decreased contractility
appear related to cAMP levels and the ratio of prostacyclin
29
to thromboxane-A2.
Cyclic-AMP is increased in varicose
vein specimens compared with normal GSVs. In addition,
the ratio of prostacyclin to thromboxane-A2 is increased
even though absolute protein levels do not di er between
normal veins and varicosities. Whether venodilation of varicosities is caused by diminished endothelin receptor levels
and responsiveness to cAMP or by a secondary e ect of
varix formation is not known. However, it is clear that with
the development of vein wall brosis, varicose veins demonstrate decreased contractile properties that probably exacerbate the development of ambulatory venous hypertension.
H I S T O R I C A L T H E O R I E S
In the twentieth century numerous theories were postulated regarding the etiology of CVI and the cause of venous
ulceration. e venous stasis, arteriovenous stula, and diffusion block theories have been disproven over time and
are discussed here for historical interest only. e etiology
for dermal skin pathology is primarily a chronic in ammatory process, and the events regulating these events are
discussedlater.
VENOUS STASISTHEORY
In 1917, John Homans published a manuscript titled “ e
Etiology and Treatment of Varicose Ulcer of the Leg,” in
30
Surgery, Gynecology, and Obstetrics.
is manuscript was
a clinical treatise on the diagnosis and management of
patients with CVI. In this manuscript Dr.Homans coined
the term “post-phlebitic syndrome” and speculated on the
cause of venous ulceration. He stated that “Overstretching
of the vein walls and destruction of the valves upon which
the mechanism principally depends bring about a degree of
surface stasis which obviously interferes with the nutrition
of the skin and subcutaneous tissues. . . . It is to be expected,
therefore that skin which is bathed under pressure in stagnant venous blood will readily form permanent, open sores
30
or ulcers.”
is statement resulted in a generation of
investigators trying to seek a causal relationship between
hypoxia, stagnant blood ow, and the development ofCVI.
e rst investigator to address the question of hypoxia
31
and CVI scienti cally was Alfred Blalock.
He obtained
venous samples from the femoral, great saphenous, and varicose veins in ten patients with CVI isolated to one limb and
compared their oxygen content with samples taken from corresponding veins in the opposite limb. Seven of the patients
had active ulcers at the time. All samples were collected in
the recumbent and standing positions. He reported that in
patients with unilateral CVI the oxygen content was higher
in the femoral vein of the a ected limb. He speculated that
this observation may be re ective of increased venous ow
rather than stagnation.
ARTERIOVENOUS FISTULATHEORY
e concept of increased venous ow in the dermal venous
plexus was expanded upon by Pratt, who reported that
increased venous ow in patients with CVI could be clini-
32
cally observed.
He attributed the development of venous
ulceration to the presence of arteriovenous connections
PATHOPHYSIOLOGY OF CHRONIC VENOUS INSUFFICIENCY • 69
and coined the term “arterial varices.” He reported that in
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a series of 272 patients with varicose veins who underwent
vein ligation, 24% had arteriovenous connections. Of the
61 patients who developed recurrences, 50% had arteriovenous communications identi ed clinically by the presence
of arterial pulsations in venous conduits. Pratt hypothesized that increased venous ow shunted nutrient- and
oxygen-rich blood away from the dermal plexus, leading to
areas of ischemia and hypoxia and resulting in venous ulceration. Pratt’s clinical observations however, have never been
con rmed with objective scienti c evidence. Experiments
with radioactively labeled microspheres have never demonstrated shunting and have therefore cast serious doubts on
the validity of this theory.
trapping theory.
36
is theory proposes that circulating
neutrophils are trapped in the venous microcirculation secondary to venous hypertension. e subsequent sluggish
capillary blood ow leads to hypoxia and neutrophil activation. Neutrophil activation leads to degranulation of toxic
metabolites with subsequent endothelial cell damage. e
ensuing heterogeneous capillary perfusion causes alterations in skin blood ow and eventual skin damage. e
problem with the leukocyte trapping theory is that neutrophils have never been directly observed to obstruct capillary ow, therefore casting doubt on its validity. However,
there is signi cant evidence that leukocyte activation plays
a major role in the pathophysiology ofCVI.
DIFFUSION BLOCKTHEORY
Hypoxia and alterations in nutrient blood ow again were
proposed as the underlying etiology of CVI in 1982 by
33
Burnand etal.
ese authors performed a study in which
skin biopsies were obtained from 109 limbs of patients with
CVI and 30 limbs from patients without CVI. Foot vein
pressures were measured in the CVI patients at rest and
a er 5, 10, 15, and 20 heel raises. Vein pressure measurements were then correlated with the number of capillaries
observed on histologic section. e authors reported that
venous hypertension was associated with increased numbers
of capillaries in the dermis of patients with CVI. Whether
the histologic sections represented true increases in capillary quantity or an elongation and distension of existing
capillaries was not answered by this study. However, in a
canine hind-limb model, the authors were able to induce
enlargement in the number of capillaries with experimen-
34
tally induced hypertension.
is important investigation
was one of the rst studies to demonstrate a direct e ect
of venous hypertension on the venous microcirculation. In
a later study, Browse and Burnand noted that the enlarged
capillaries observed on histologic examination exhibited
pericapillary brin deposition and coined the term “ brin
35
ey speculated that venous hypertension led to
cu .”
widening of endothelial gap junctions with subsequent
extravasation of brinogen leading to the development of
brin cu s. ese authors theorized that the cu s acted as a
barrier to oxygen di usion and nutrient blood ow, resulting in epidermal cell death. Although pericapillary cu s do
exist, it has never been demonstrated that they act as a barrier to nutrient ow or oxygen di usion.
LEUKOCYTE ACTIVATION
Dissatisfaction with the brin cu theory and subsequent
observations of decreased circulating leukocytes in blood
samples obtained from the GSVs in patients with CVI led
Coleridge Smith and colleagues to propose the leukocyte
ROLE OF LEUKOCYTE
ACTIVATION AND FUNCTIONAL
STATUSINCVI
In 1988, omas etal. reported that 24% fewer white cells
le the venous circulation a er a period of recumbency in
37
patients with CVI as compared with normal patients.
ey
studied three groups of ten patients each. Group1 consisted
of patients with no signs of venous disease. Group2 were
patients with uncomplicated primary varicose veins, and
Group3 were patients with long-standing CVI as determined by Doppler ultrasonography, strain-gauge plethysmography, and foot volumetry. Patients had the GSV
cannulated just above the medial malleolus. Venous samples
were obtained at various time points with patients in the
sitting and supine position. Samples were then placed in
an automated cell counter, and the number of leukocytes
and erythrocytes determined. e ratios of white cells to
red cells at the various time points were then compared.
e authors reported that with leg dependency, packed
cell volume signi cantly increased in patients with CVI
as compared with normal controls, whereas patients with
primary varicose veins showed no di erence from controls.
ey also noted that the relative number of white cells were
signi cantly decreased compared to control and primary
varicose vein patients (28% vs. 5%, p < 0.01). e authors
concluded that the decrease in white cell number was due to
leukocyte trapping in the venous microcirculation secondary to venous hypertension. ey further speculated that
while trapped, leukocytes may be activated and release toxic
metabolites, causing damage to the microcirculation and
the overlying skin. ese important observations were the
rst to implicate abnormal leukocyte activity in the pathophysiology ofCVI.
e importance of leukocytes in the development of der-
38
mal skin alterations was emphasized by Scott etal.
ese
authors obtained punch biopsies from patients with primary varicose veins, patients with lipodermatosclerosis, and
patients with lipodermatosclerosis and healed ulcers, and
determined median number of white blood cells (WBCs)
70 • BASIC CONSIDERATIONS
per high power eld (40× magni cation) in each group. No
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patients with active ulcers were included, and no attempt
to identify the type of leukocytes was made. e authors
reported that in patients with primary varicose veins, lipodermatosclerosis, and healed ulceration there was a median
2
of 6, 45, and 217 WBCs per mm
, respectively. is study
demonstrated that with clinical disease progression and
increasing severity of CVI, there was a progressive increase
in the number of leukocytes in the dermis of CVI patients.
e types of leukocytes involved in dermal venous stasis skin changes are controversial. In a study performed
by Wilkerson et al., skin biopsies were obtained from
twenty-three patients who required surgical ligation, strip-
39
ping, and/or avulsion for their varicose veins.
e condition of the skin was recorded as liposclerotic, eczematous, or
normal. Lipodermatosclerosis was de ned clinically as palpable induration of the skin, and subcutaneous tissues and
eczema as visible erythema with scaling of the skin. Using
immunohistochemical techniques, the authors stained for
leukocyte-speci c cell surface markers and reported that
macrophages and lymphocytes were the predominant leukocytes observed in this patient population. Neutrophils
and B lymphocytes rarely were observed. T lymphocytes
and macrophages were predominantly observed perivascularly and in the epidermis. However, Pappas etal. performed
a quantitative morphometric assessment of the dermal
microcirculation using electron microscopy and reported
that macrophages and mast cells were the predominant
cells observed in patients with CVI dermal skin changes.
40
Furthermore, lymphocytes were never observed. is discrepancy may re ect the types of patients that were studied.
Wilkerson etal. biopsied patients with erythematous and
eczematous skin changes, whereas Pappas predominantly
evaluated older patients with dermal brosis. Patients with
eczematous skin changes may have an autoimmune component to their CVI, whereas patients with dermal brosis
may re ect changes consistent with chronic in ammation
and altered tissue remodeling.
Given the predominant role of leukocytes in CVI
pathology, there has been great interest in the activation
state and functional status of leukocytes in CVI patients.
Pappas etal. explored the hypothesis that circulating leukocytes in CVI patients were in an altered state of activation and therefore may be involved in leukocyte-mediated
injury. ey measured the expression of cell surface activation markers of circulating leukocytes using uorescence
41
ow cytometry.
Relative to normal individuals, patients
with chronic venous stasis ulcers had a decreased expression
of the CD3+/DR+ and CD3+/CD38+ markers on T lymphocytes and an increased expression of CD14+/CD38+
markers on monocytes. Circulating neutrophils demonstrated no evidence of activation.
Although Pappas etal. identi ed a population of circulating cells demonstrating altered activation markers, their
results did not test the functional status of these cells. In
a follow-up study, Pappas etal. tested the hypothesis that
circulating mononuclear cells in CVI patients were dysfunctional by challenging monocytes with test mitogens.
Lymphocyte and monocyte cel l function was measured as the
degree of proliferation in response to a mitogenic challenge.
Fi y patients were separated into four groups:Group 1,
fourteen patients with normal limbs; Group2, ten patients
with class 2 CVI (stasis dermatitis only); Group3, een
patients with active venous ulcers; Group4, eleven patients
with healed venous ulcers and current evidence of lipodermatosclerosis. Systemically circulating lymphocytes and
monocytes were obtained by antecubital venipuncture
from Groups 1–4. Cells were cultured in the presence of
staphylococcal enterotoxins (SEs) A, B, C
, D, and E (mito-
1
gens) and PHA (phytohemagglutinin), a control mitogen.
Proliferative responses to PHA indicated that lymphocytes and monocytes from CVI patients were not globally
depressed. However, patients in Group2 did not exhibit
the same degree of proliferation to PHA as did Groups
1, 3, and 4.Di erences in proliferative responses between
Groups 2 and 1 (44.38± 43.9 vs. 118.87± 27.1, p < 0.05)
and Groups 2 and 3 (44.38± 43.9 vs. 105.95± 60.99, p
< 0.05) were signi cant. Challenges with staphylococcal
enterotoxin Aand B revealed signi cant diminution of proliferative responses in Groups 2 (42.73± 11.55, p < 0.05)
and 3 (45.57± 9.1, p < 0.05) and Groups 3 (36.81± 6.9, p
< 0.05) and 4 (35.04± 7.5, p < 0.05), compared with SEA
controls (68.68± 9.9) and SEB controls (66.25± 13.56),
respectively. A trend toward diminished cellular function
with progression of CVI was observed with staphylococcal
enterotoxins B, C
, D, and E, strongly suggesting biologic
1
signi cance. Furthermore, patients with lipodermatosclerosis and a history of healed ulcers uniformly exhibited the
poorest proliferative responses. is study indicated that
deterioration of mononuclear cell function was associated
with CVI and suggested that lymphocyte and monocyte
function diminished with clinical disease progression. e
authors speculated that the decreased capacity for mononuclear cell proliferation in response to various challenges
may manifest itself clinically as poor and prolonged wound
healing.
T H E V E N O U S
MICROCIRCULATION
Numerous investigations have attempted to evaluate the
40,43–46
microcirculation of patients with CVI.
e majority of these investigations were qualitative descriptions of
vascular abnormalities, which lacked uniformity of biopsy
sites and patient strati cation. Prior to 1997 it was widely
accepted that endothelial cells from the dermal microcirculation appeared abnormal, contained Weibel-Palade bodies,
were edematous, and demonstrated widened interendothe-
45
lial gap junctions.
Based on these descriptive observations
42
PATHOPHYSIOLOGY OF CHRONIC VENOUS INSUFFICIENCY • 71
it was assumed that the dermal microcirculation of CVI
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patients have functional derangements related to permeability and ulcer formation. It was not until 1997 that a
quantitative morphometric analysis of the dermal microcir-
40
culation was reported.
e objectives of this investigation
were to quantify di erences in endothelial cell structure
and local cell type with emphasis on leukocyte cell type and
their relationship to arterioles, capillaries, and postcapillary venules (PCVs). Variables assessed were number and
types of leukocytes, endothelial cell thickness, endothelial vesicle density, interendothelial junctional width, cu
thickness, and ribosome density. irty- ve patients had
two 4-mm punch biopsies obtained from the lower calf
(gaiter region) and lower thigh. Patients were separated
into one of four groups according to the 1995 ISCVS/SVS
(International Society for Cardiovascular Surgery/Society
5
for Vascular Surgery) CEAP classi cation.
Group1 consisted of ve patients with no evidence of venous disease.
Skin biopsies from these patients served as normal controls.
Groups 2 through 4 consisted of patients with CEAP Class
4 (n=11), Class 5 (n=9), and Class 6 (n=10)CVI.
ENDOTHELIAL CELL
CHARACTERISTICS
No signi cant di erences were observed in endothelial
cell thickness of arterioles, capillaries, and PCVs from
40
either gaiter or thigh biopsies.
ualitatively, endothelial
cells appeared metabolically active. Many nuclei exhibited a euchromatic appearance, implying active mRNA
transcription. In most instances ribosome numbers were
so abundant that they exceeded the resolution capacity
of the image analysis system and could not be quanti ed.
e prominence in ribosome content and the euchromatic
appearance of the endothelial cell nucleus strongly suggested active protein production. No signi cant di erences
in vesicle density were observed in gaiter biopsies between
groups. Class 6 patients exhibited an increased number of
vesicles in arterioles and PCV endothelia from thigh biopsies but did not di er compared with gaiter biopsies. Mean
interendothelial junctional width varied within a normal
range of 20–50nm. Signi cantly widened interendothelial
gap junctions were not observed and thus con icted with
45
the reports of Wenner et al.
Mean basal lamina thickness di ered signi cantly at the capillary level in both gaiter and thigh biopsies. Di erences were most pronounced
in patients with Class 4 disease. ese data indicated that
endothelial cells from the dermal microcirculation of CVI
patients were far from normal. ey demonstrated increased
metabolic activity suggestive of active cellular transcription
and protein production. Most surprising was the observation of uniformly tight gap junctions. Previously these gap
junctions were reported to be as wide as 180nm, and it was
assumed that these widened junctions were responsible for
macromolecule extravasation and edema formation.
33,45
Pappas et al. suggested that alternate methods for tissue
edema such as increased transendothelial vesicle transport,
formation of transendothelial channels, and alterations in
the glycocalyx lining the junctional cle may be involved in
40
CVI edema and macromolecule transport.
TYPES AND DISTRIBUTION OF
LEUKOCYTES
e most striking di erences in cell type and distribution were observed with mast cells and macrophages (see
Figure8.3). In both gaiter and thigh biopsies, mast cell numbers were two to four times greater than control in Class
4 and 5 patients around arterioles and PCVs (p < 0.05).
Class 6 patients demonstrated no di erence in mast cell
number compared to controls. Mast cell numbers around
capillaries did not di er across groups in either gaiter or
thigh biopsies. Macrophages demonstrated increased numbers in Class 5 and 6 patients around arterioles and PCVs,
respectively (p < 0.05). Di erences in macrophage numbers around capillaries were observed primarily in Class 4
patients in both gaiter and thigh biopsies. Surprisingly, lymphocytes, plasma cells, and neutrophils were not present in
the immediate perivascular space. Fibroblasts were the most
common cells observed in both gaiter and thigh biopsies.
It was speculated that mast cells and macrophages may
function to regulate tissue remodeling resulting in dermal
Figure8.3 Electron micrograph (Mag 4300×) of mast cells (MC),
macrophages (MP) and broblast (F)surrounding a central capillary
from dermal biopsy of a patient with CEAP class 4 chronic venous
insu ciency.
72 • BASIC CONSIDERATIONS
brosis. 40 e mast cell enzyme chymase is a potent activa-
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tor of matrix metalloproteinase-1 and -3 (collagenase and
47–49
stromelysin).
In an in vitro model using the human mast
cell line HMC-1, these cells were reported to spontaneously
adhere to bronectin, laminin, and collagen types I and
III, all components of the perivascular cu (see later).
49
Chymase also causes release of latent transforming growth
factor-beta 1 (TGF-β
) secreted by activated endothelial
1
cells, broblasts, and platelets from extracellular matri-
50
Release and activation of TGF-,β 1 initiate a cascade of
ces.
events in which macrophages and broblasts are recruited
to wound healing sites and stimulated to produce broblast
mitogens and connective tissue proteins, respectively.
Mast cell degranulation leading to TGF-β
activation and
1
51
macrophage recruitment may explain why decreased mast
cell and increased macrophage numbers were observed in
Class 6 patients. Macrophage migration, as evidenced by
the frequent appearance of cytoplasmic tails in perivascular
macrophages, further substantiates the concept of in ammatory cytokine recruitment (see Figure8.4).
EXTRACELLULAR MATRIX
ALTERATIONS
Once leukocytes have migrated to the extracellular space
they localize around capillaries and postcapillary venules.
e perivascular space is surrounded by extracellular matrix
(ECM) proteins and forms a perivascular cu . Adjacent to
these perivascular cu s and throughout the dermal interstitium is an intense and disorganized collagen deposition.
33,40
Perivascular cu s and the accompanying collagen deposition are the sine qua non of the dermal microcirculation in
CVI patients (see Figure8.4). e perivascular cu originally was thought to be the result of brinogen extravasation
Migrating
Pericapillary
Cu
Fibroblast
Postcapillary
Venule
Figure8.4 Electron micrograph (Mag 4300×) of a well-developed
perivascular cu in close proximity to a broblast in a patient with
CEAP class 6 chronic venous insu ciency. Long arrow points to
macrophages that appear to be entering a lymphaticlumen.
Macrophages
Lymphatic
5
and erroneously referred to as a brin cu .
It is now known
that the cu is a ring of ECM proteins consisting of collagen
types Iand III, bronectin, vitronectin, laminin, tenascin,
52
and brin.
e role of the cu and its cell of origin is not
completely understood. e investigation by Pappas etal.
suggested that the endothelial cells of the dermal microcirculation were responsible for cu formation.
40
e cu was
once thought to be a barrier to oxygen and nutrient di usion; however, recent evidence suggests that cu formation
is an attempt to maintain vascular architecture in response
53
to increased mechanical load.
Although perivascular cu s
may function to preserve microcirculatory architecture,
several pathologic processes may be related to cu formation. Immunohistochemical analyses have demonstrated
TGF-β
cular cu s.
and α 2 -macroglobulin in the interstices of perivas-
1
54
It has been suggested that these “trapped” molecules are distributed abnormally in the dermis, leading to
altered tissue remodeling and brosis. Cu s may also serve
as a lattice for capillary angiogenesis, explaining the capillary tortuosity and increased capillary density observed in
the dermis of CVI patients.
PATHOPHYSIOLOGY OF STASIS
DERMATITIS AND DERMAL
FIBROSIS
e mechanisms modulating leukocy te activation, broblast
function, and dermal extracellular matrix alterations have
been the focus of investigation in the 1990s. CVI is a disease
of chronic in ammation due to a persistent and sustained
injury secondary to venous hypertension. It is hypothesized
that the primary injury is extravasation of macromolecules
(i.e., brinogen and α
(RBCs) into the dermal interstitium.
-macroglobulin) and red blood cells
2
33,34,44,45,54
RBC degradation products and interstitial protein extravasation
are potent chemoattractants and presumably represent
the initial underlying chronic in ammatory signal responsible for leukocyte recruitment. It has been assumed that
these cytochemical events are responsible for the increased
expression of ICAM-1 (intercellular adhesion molecule-1)
on endothelial cells of microcirculatory exchange ves-
39,55
sels observed in CVI dermal biopsies.
ICAM-1 is the
activation-dependent adhesion molecule utilized by macrophages, lymphocytes, and mast cells for diapedesis. As
stated earlier, all these cells have been observed by immunohistochemistry and electron microscopy in the interstitium
39,40
of dermal biopsies.
CYTOKINE REGULATION AND
TISSUE FIBROSIS
Leukocyte recruitment, ECM alterations, and tissue brosis
are characteristic of chronic in ammatory diseases caused by
PATHOPHYSIOLOGY OF CHRONIC VENOUS INSUFFICIENCY • 73
alterations in TGF-β 1 gene expression and protein produc-
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tion. To determine the role of TGF-β
in CVI, dermal biop-
1
sies from normal patients and CEAP Class 4, 5, and 6 CVI
patients were analyzed for TGF-β
production, and cellular location.
for TGF-β
gene expression was performed on twenty-four
1
gene expression, protein
1
56
uantitative RT-PCR
skin biopsies obtained from twenty-four patients. Patients
were separated into four groups according to the ISCVS/
SVS classi cation for CVI:normal skin (n =6), CEAP
Class 4 (n=6), CEAP Class 5 (n=5), and CEAP class
6 (n= 7). TGF-β
gene transcripts for controls, Class 4,
1
5, and 6 patients were 7.02± 7.33, 43.33± 9.0, 16.13±
–14
7.67, and 7.22± 0.56 x 10
tively. e di erences in TGF-β
moles/g total RNA, respec-
gene expression in Class
1
4 patients was signi cantly elevated compared with control
56
and Class 5 and 6 patients (p < 0.05).
An additional 38
patients had 54 biopsies from the lower calf (LC) and lower
thigh (LT) analyzed for TGF-β
e amounts of active TGF-β
protein concentration.
1
in picograms/gram (pg/g)
1
of tissue from LC and LT biopsies compared to normal skin
biopsies were as follows:Normal skin (<1.0 pc/g), Class 4
(LC, 5061± 1827; LT 317.3± 277), Class 5 (LC, 8327±
3690; LT 193± 164), and Class 6 (LC, 5392± 1800; LT,
117± 61; see Figure8.5). Di erences between normal skin
and Class 4 and 6 patients were signi cant (p < 0.05 and p
< 0.01, respectively). No di erences between Class 4, 5, and
6 patients were observed. Di erences between LC and LT
within each CVI group were signi cant (Class 4, p < 0.003,
Class 5, p < 0.008, Class 6, p < 0.02). ese data demonstrate
that in areas of clinically active CVI, increased amounts
of active TGF-β
Furthermore, active TGF β
15,000
10,000
are present compared with normal skin.
1
Results: Active TGF-β1 Protein
Levels From CVI Dermal Skin
LC=Lower Calf
LT=Lower Thigh
protein concentrations of
1
Biopsies
#
biopsies from the LT did not di er from normal skin demonstrating a regionalized response to injury.
56
Immunohistochemistry and immunogold labeling
experiments were performed to identify the sources of
active TGF-β
protein production. Immunohistochemistry
1
of normal skin and ipsilateral thigh biopsies of CVI patient
demonstrated mild TGF-β
in the basal layer of the epider-
1
mis. e dermis demonstrated few capillaries, ordered collagen architecture, and no interstitial leukocytes. CVI dermal
biopsies from areas of clinically active disease demonstrated
staining of the basal layer of the epidermis, interstitial leukocytes, and broblasts. Many perivascular leukocytes
demonstrated positive staining of intracellular granules and
appeared morphologically similar to previously reported
56
mast cells (see Figures8.3 and 8.6).
Numerous capillaries
with perivascular cu s were observed; however, cu s did
not stain positively for TGF-β
. 56 is study con icts with
1
the observations reported by Higley et al. in which they
reported positive TGF-β
an absence of TGF-β
ulcer compared to healing donor skin gra sites.
cluded that TGF-β
staining in perivascular cu s and
1
in the provisional matrix of the venous
1
was therefore abnormally “trapped” in
1
54
ey con-
the perivascular cu and therefore unavailable for normal
granulation tissue development. Di erences between the
two studies may relate to biopsy site selection. Higley etal.
biopsied chronic, nonhealing venous ulcer edges and ulcer
bases, whereas patients with active ulcers in the study by
Pappas etal. were biopsied 5 to 10cm away from an active
ulcer. erefore, the former study re ects the biology of
chronic wound healing, and our data suggest active tissue
remodeling in response to a chronic injury stimulus.
Immunogold labeling con rmed the presence of TGF-β
1
in dermal leukocytes. Positive labeling of gold particles
similarly were observed in collagen brils of the ECM. is
observation may explain why the molecular regulation of
TGF-β
in CVI patients demonstrates di erential gene and
1
protein production according to disease classi cation. As
5,000
1 levels in pg/gm of tissue
β
TGF-
Figure8.5 Active TGF-β
and 6 patients compared with controls and ipsilateral thigh biopsies.
Con-Control patients without venous disease, LT-Ipsilateral thigh,
LC-Ipsilateral diseasedskin.
*
0
CON C4 LC C4 LT C5 LC C5 LT C6 LC C6 LT
* Control vs Class 4 and 6 (p
# LC vs LT biopsies within each class (p
*, #
#
CVI Patient Classification
≤
0.05)
levels indicating increased levels in class 4, 5,
1
*, #
#
≤
0.02)
74 • BASIC CONSIDERATIONS
#
Figure8.6 Immunohistochemistry (Mag 575×) of dermal skin biopsy
demonstrating transforming growth factor-β
arrow) in leukocytes surrounding a perivascular cu and leukocytes
migrating through a perivascular cu (short arrow).
positive granules (long
1
stated earlier, the gene expression of TGF-β 1 was increased in
https://t.me/med1917
Class 4 patients only, and the protein production essentially
was increased in Class 4, 5, and 6 patients. ese di erences
may be related to disease severity and the pluripotential
responses of TGF-β
TGF-β 1 can have inhibitory and stimu-
r
latory e ects that are primarily dependent on local concentration, cell source, and surrounding ECM. In the study by
Pappas etal., Class 4 patients were younger than the other
study groups, never experienced an episode of venous stasis
ulceration, and clinically demonstrated less dermal tissue
brosis. TGF-β
in these patients therefore may be involved
1
in limiting the response to injury. Indeed, one could speculate that early on in the disease process, a low-grade production of TGF-β
is a normal wound-healing response and may
1
serve to prevent the onset and development of tissue brosis.
With continued and prolonged exposure, an imbalance in
tissue remodeling in patients with Class 5 and 6 disease clinically manifests itself as dermato brosis. Apathologic e ect
of increased ECM deposition is an alteration in the storage
57
and release of growth factors.
e latent form of TGF-β 1
is secreted from cells bound to one of three latent TGF-β
binding proteins (LTBPs). Once secreted, LTBPs mediate
binding of latent TGF-β
of TGF-β
is mediated by multiple serine proteinases includ-
1
ing plasmin, mast cell chymase, and leukocyte elastase.
to matrix proteins. Matrix release
1
50,58–60
An increase in the number of mast cells and circulating
leukocyte elastase have been reported previously in CVI
40,61
patients.
e increase in active TGF-β 1 observed in Class
5 and 6 patients therefore may result from ECM release of
latent TGF-β
, resulting in tissue brosis. is hypothesis is
1
consistent with the demonstration of immunogold labeling
to collagen brils in the ECM of CVI patients. e modulation of TGF-β
release from the ECM may therefore provide
1
a faster means of signal transduction than simple control of
gene expression, and therefore may explain the sustained
increase of TGF-β
in Class 5 and 6 patients in the absence
1
of increased gene expression. is study did not demonstrate
increased TGF-β
staining in the ECM by ICC because the
1
primary antibody used was speci c only for active TGF-β
and therefore may have missed latency associated peptide
(LAP) and LTBP associated TGF-β
.
1
e distribution and location of several other growth factors in the skin of CVI patients have also been investigated.
Peschen etal. reported on the role of platelet-derived growth
factor receptor alpha and beta (PDGFR-α and -β) and vas-
62
cular endothelial growth factor (VEGF).
Skin biopsies
from 30 patients were separated into ve groups:Group1,
patients with reticular veins; Group 2, venous eczema;
Group3, skin pigmentation; Group4, lipodermatosclerosis; and Group5, patients with active leg ulcers; with a total
of six patients in each group. Biopsies were studied with
immunohistochemistry and the degree of immunoreactivity assessed with a scoring system by two blinded reviewers.
Peschen etal. reported that PDGFR-α and -β and vascular
endothelial growth factor (VEGF) expression was strongly
increased in the stroma of CVI patients with eczema and
active ulcers compared with patients with reticular veins
62
and pigmentation changes only.
To a lesser degree, patients
with lipodermatosclerosis demonstrated immunoreactivity to PDGFR-α and -β and VEGF as well. PDGFR-α
and -β expression was elevated considerably in the capillaries and surrounding broblasts and in ammatory cells of
venous eczema patients. In addition, immunoreactivity was
increased in dermal broblasts, smooth muscle cells, and
vascular cells of lipodermatosclerosis patients compared
with patients with reticular veins only. e greatest expression of PDGFR-α and -β was observed in mesenchymal
cells and vascular endothelial cells of patients with active
venous ulcers. VEGF immunoreactivity correlated with disease severity. VEGF positive capillary endothelial cells and
pericapillary cells increased in patients with venous eczema,
lipodermatosclerosis, and active venous ulceration, respectively. In a subsequent investigation, these authors reported
that with progression of CVI dermal pathology the endothelial cell adhesion molecules intercellular and vascular
adhesion molecules (ICAM-1, VCAM-1) and their corre-
1
sponding leukocyte ligands LFA-1 and VLA-4 were upreg-
55
ulated on leukocytes and endothelial cells.
Based on these
observations, the authors speculated that leukocyte recruitment, capillary proliferation, and interstitial edema in CVI
patients may be regulated through PDGF and VEGF by
upregulation of adhesion molecules leading to leukocyte
recruitment, diapedesis, and release of chemical mediators.
In summary, these investigations indicate that progression of CVI dermal pathology is mediated by a cascade of
in ammatory events. Venous hypertension causes extravasation of macromolecules like brinogen and red blood cells
that act as potent in ammatory mediators. ese mediators
cause an upregulation of adhesion molecules and the expression of growth factors like PDGF and VEGF, which result
in leukocyte recruitment. Monocytes and mast cells travel to
the site of injury, which activate or release TGF-β
and proba-
1
bly other undiscovered chemicals as well. What e ect growth
factor binding has on broblast and endothelial cell function
1
has been the focus of numerous investigations in the1990s.
DERMAL FIBROBLAST
FUNCTION
Several studies have reported aberrant phenotypic behavior
of broblasts isolated from venous ulcer edges when compared to broblasts obtained from ipsilateral thigh biopsies
of normal skin in the same patients. Hasan etal. compared
the ability of venous ulcer broblasts to produce αI procollagen mRNA and collagen a er stimulation with TGF-β
ese authors were not able to demonstrate di erences in
αI procollagen mRNA levels a er stimulation with TGF-β
between venous ulcer broblasts and normal broblasts
(control) from ipsilateral thigh biopsies. However, collagen
55
. 63
1
1
PATHOPHYSIOLOGY OF CHRONIC VENOUS INSUFFICIENCY • 75
production was increased by 60% in a dose-dependent
https://t.me/med1917
manner in controls, whereas venous ulcer broblasts were
unresponsive. is unresponsiveness was associated with a
four-fold decrease in TGF-β
report, Kim etal. indicated that the decrease in TGF-β
type II receptors. In a follow-up
1
type
1
II receptors was associated with a decrease in phosphorylation of the TGF-β
well as p42/44 mitogen activated protein kinases.
receptor substrates SMAD 2 and 3 as
1
64
Asimilar investigation reported a decrease in collagen production
from venous ulcer broblasts and similar amounts of bro-
65
nectin production when compared to normal controls.
Fibroblast responsiveness to growth factors was further
66
delineated by Stanley etal.
ese investigators characterized the proliferative responses of venous ulcer broblasts
when stimulated with basic broblastic growth factor
(bFGF), epidermal growth factor (EGF), and interleukin
1-β (IL-1β). In their initial study, they reported that venous
ulcer broblast growth rates were markedly suppressed when
stimulated with bFGF, EGF, and IL-1β. In a follow-up investigation these authors noted that the previously observed
67
growth inhibition could be reversed with bFGF.
Lal etal.
reported that the proliferative responses of CVI broblasts
to TGF-β
correlated with disease severity. 68 Fibroblasts
1
from patients with CEAP Class 2 and 3 disease retain their
agonist-induced proliferative capacity. Class 4 and 5 broblasts demonstrated diminished agonist-induced proliferation, whereas Class 6 (venous ulcer broblasts) did not
proliferate a er TGF-β
stimulation, con rming the obser-
1
vations made by the previous investigators. Phenotypically,
venous ulcer broblasts appeared large and polygonal with
varied nuclear morphologic features, whereas normal broblasts appeared compact and tapered with well-de ned
nuclear morphologic features. Venous ulcer broblasts
appeared morphologically similar to broblasts undergoing
cellular senescence. erefore, the blunted growth response
of CVI venous ulcer broblasts appears related to develop-
66,69
ment of cellular senescence.
Other characteristics of senescent cells are an overexpression of matrix proteins such as bronectin (cFN) and
enhanced activity of β-galactosidase (SA-β-Gal). In an
evaluation of seven patients with venous stasis ulcers, it
was noted that a higher percentage of SA-β-Gal positive
cells in venous ulcers compared to normal controls (6.3%
67
vs. 0.21%, p < 0.0.6).
It was also reported that venous
ulcer broblasts produced one to four times more cFN by
69
Western blot analysis compared to controls.
ese data
support the hypothesis that venous ulcer broblasts phenotypically behave like senescent cells. However, senescence is
probably the end manifestation of a wide spectrum of events
that lead to proliferative resistance and cellular dysfunction.
Telomeres and telomerase activity are the sine qua non of
truly senescent cells. To date, there are no reported studies indicating an abnormality in CVI broblast telomere
or telomerase activity. Absent these investigations, the true
role of senescence in CVI remains ill-de ned.
ROLE OF MATRIX
METALLOPROTEINASES AND
THEIR INHIBITORSINCVI
e signaling event responsible for the development of
a venous ulcer and the mechanisms responsible for prolonged wound healing are poorly understood. Wound
healing is an orderly process that involves in ammation,
re-epithelialization, matrix deposition, and tissue remodeling. Tissue remodeling and matrix deposition are processes
controlled by matrix metalloproteinases (MMPs) and tissue
inhibitors of matrix metalloproteinases (TIMPs). In general,
MMPs and TIMPs are not constitutively expressed. ey are
induced temporarily in response to exogenous signals such
as various proteases, cytokines or growth factors, cell-matrix
interactions, and altered cell-cell contacts. TGF-β
is a
1
potent inducer of TIMP-1 and collagen production and
inhibitor of MMP-1 through regulation of gene expression
and protein synthesis. Several studies have demonstrated
that prolonged and continuous TGF-β
production causes
1
tissue brosis by stimulating ECM production and inhibiting degradation by a ecting MMP and TIMP production.
Alterations in MMP and TIMP production may similarly
modulate the tissue brosis of the lower extremity in CVI
patients. Several investigators have reported that the gelatinases MMP-2 and -9 as well as TIMP-1 are increased in the
exudates of patients with venous ulcers compared to acute
70–72
wounds.
However, analyses of biopsy specimens have
demonstrated variable results. Herouy etal. reported that
MMP-1 and -2 and TIMP-1 are increased in patients with
73
lipodermatosclerosis compared with normal skin.
In a subsequent investigation, biopsies from venous ulcer patients
were found to have increased levels of the active form of
74
MMP-2 compared with normal skin
as well as increased
immunoreactivity to EMMPRIN (extracellular inducer of
MMP), MT1-MMP (membrane type 1), and MT2-MMP
in the dermis and perivascular regions of venous ulcers.
75
Saito et al. were unable to identify di erences in overall
MMP-1, -2, and -9 and TIMP-1 protein levels or activity in CVI patients with CEAP Class 2 through 6 disease
76
compared with normal controls or CVI groups.
However,
within a clinical class, MMP-2 levels were elevated compared
with MMP-1, and -9 and TIMP-1 in patients with Class 4
and Class 5 disease. ese data indicate that active tissue
remodeling is occurring in patients with CVI. Which matrix
metalloproteinases are involved and how they’re activated
and regulated are currently unclear. It appears that MMP-2
may be activated by urokinase plasminogen activator (uPA).
Herouy et al. observed increased uPA and urokinase-type
plasminogen activator receptor (uPAR) mRNA and protein
levels in patients with venous ulcers compared to normal
77
e elevated levels of active TGF-β 1 in the dermis
skin.
of CVI patients suggests a regulatory role for TGF-β
1
in
MMP and TIMP synthesis and activity. However, there is
currently no direct evidence indicating such a relationship.
76 • BASIC CONSIDERATIONS
C O N C L U S I O N
https://t.me/med1917
e mechanisms regulating varicose vein development and
the subsequent dermal skin sequelae caused by chronic
ambulatory venous hypertension only recently have been
investigated. It is clear that varicose vein formation has a
genetic component that is linked to environmental stimuli.
Susceptible patients develop vein wall brosis and loss of
valvular competence that leads to venous hypertension. e
transmission of high venous pressures to the dermal microcirculation causes extravasation of macromolecules and red
blood cells that serve as the underlying stimulus for in ammatory injury. Activation of the microcirculation results
in cytokine and growth factor release leading to leukocyte
migration into the interstitium. At the site of injury, a host
of in ammatory events is set into action. TGF-β
to be a primary regulator of CVI induced injury. TGF-β
appears
1
1
secretion from leukocytes with subsequent binding to dermal broblasts is associated with intense dermal brosis
and tissue remodeling. In addition, decreased TGF-β
type
1
II receptors on venous ulcer broblasts are associated with
diminished broblast proliferation. Fibroblast proliferation diminishes with disease progression, ultimately leading
to senescence and poor ulcer healing. In addition, increases
in MMP-2 synthesis appear to increase tissue remodeling
and further impede ulcer healing. As our understanding
of the underlying cellular and molecular mechanisms that
regulate CVI and ulcer formation increase, therapeutic
interventions for treatment and prevention will ultimately
follow.
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