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80 Venous ulcer formation and healing atcellularlevels
https://t.me/med1917
GP
coupled
R
PLA
N-Cadherin
expression
2
Cx43
Stress, inflammation
TGF
TGF-βR
Ser Thr
Kin
PL
Smad 2/3
AA PGE
pRb-PO
Fas L, TNF, IL
Death R
Ask 1
Tak 1
Map2k6
Smad 4
2
pRb
4
Bcl-2
p53
GP
Rac
Hormones
R
1
AC
cAMP p44/p42
MEKK
JNK/SAPK
FGF, EGF, PDGF
Tyr Kin
Ras
Kinase activation
Signal transduction
p38
Arrest of DNA
Transcriptional factors
Repression of genes
E2F, Id, c-fos
Genes not expressed
Cyclin A, Cdc2, DHFR, TK,
DNA poly-α, PCNA
GF R
PI3K
Raf 1
Hormone, EGF, PDGF
GF R
2
GP
PIP
2
p44/p42
Blocked-
attenuated
response
PLC
IP3 + DAG
CDK
membrane
Nucleus
Cell
PMA, TMA
PKC
Raf 1
p21
p16
XR
UV
H2O
Drugs
Toxins
Disease
2
Figure 7.3 Signaling pathways leading to inhibition of DNA transcription and cell proliferation: senescence phenotype in
venous ulcer fibroblasts. Schematic of the venous ulcer fibroblast signaling pathways for the regulation of cell proliferation
and the pathways leading to growth attenuation and DNA inhibition rendering these cells with a senescent phenotype.
Growth factors, hormones, and cytokines bind to cell surface receptors (TGF, death, Hormone R1, and growth factors), and
various noxious stimuli (inflammation, radiation, and stress) and phorbol esters (phorbol-12-myristate 13-acetate [PMA] and
12-tetradecanoate phorbol 13-acetate [TPA]) have direct or indirect effects on receptors. Following receptor activation,
signal pathways lead to protein phosphorylation and the production of secondary messengers involving various membraneassociated proteins: tyrosine kinase (Tyr Kin), serine threonine kinase (Ser Thr Kin), G-protein (GP), adenylyl cyclase (AC), Ras,
Rac, phosphatidylinositol 3-kinase (PI3K), phospholipase C (PLC), and protein kinase C (PKC). The Ras (a GDP/GTP-activated
protein)-dependent pathway activates the kinase cascade, activating Raf, MEK, and MAPK (ERK1/2 and p44/p42), and the
Ras-independent pathway leads to the activation of PKC, Raf, and MAPK for the activation of transcription factors (Elk-1,
c-Myc, CREB, and Sap-1) and DNA transcription and proliferation. Phosphoinositol 4,5-biphosphate (PIP
in which PLC forms the secondary messengers inositol 1,4,5-triphosphate (IP
) and 1,2-diacylglycerol (DAG). In turn, DAG is
3
) is the substrate
2
important in activating PKC by membrane translocation. The ligand-stimulated TGF-βR receptor complex causes phosphorylation of the Smad complex (Smad 2/3–Smad 4) and translocates to the nucleus, binding to transcription factors and leading to gene activation. Stimulation of the death receptors by cytokines and stress activates the Rac, Ask, and Tak pathways,
leading to kinase activity (MEKK and Map2k6) and phosphorylation of p38 and JNK/SAPK kinase, leading to transcription
factor activation (c-Jun, ATF-2, Elk-1, Sap-1, and CHOP), which brings about growth arrest and apoptosis. Note that active
Ras can also activate p38 and JNK (not shown in the diagram). Senescent cells and venous ulcer fibroblasts (senescent-like
phenotype) have attenuated responses to signal transduction (=), leading to inhibition of DNA synthesis. Arrest of DNA
synthesis is also a result of increased metabolites of phospholipids (PLs) by the action of phospholipase A2 (PLA
ing elevated levels of arachidonic acid (AA) and prostaglandin E
(PGE2), and by the inhibition of cyclin-dependent protein
2
kinases (CDKs) by the overexpression of p21 and p16, causing underphosphorylation of pRb (i.e., decreased pRb-PO
), produc-
2
) and
4
consequently inhibition of the gene expression that is necessary for DNA replication. Unlike senescent cells, venous ulcer
fibroblasts, despite having fewer mitogenic receptors, utilize the MAPK pathway (elevated ERK 1/2 and p44/p42) and are
able to respond to growth factors (bFGF) and downregulate negative proliferative proteins and kinases (p21 and p38) in
order to increase proliferation. Dihydrofolate reductase (DHFR), thymidine kinase (TK), DNA polymerase-α (DNA polyα), and
the cofactor proliferating cell nuclear antigen (PCNA). Dashed arrows indicate a pathway; solid up or down arrows indicate
whether that compound is overexpressed or underexpressed, respectively; and double solid bars indicate a blocked/attenuated response.
7.8 Wound fluid environment andMMPs 81
https://t.me/med1917
dierentiation pathways, and may in part be responsible
for impaired VLU healing.
68
7.8 WOUND FLUID ENVIRONMENT
ANDMMPs
7.8.1 Venous ulcer wound fluid
e venous ulcer microenvironment consists of dermal broblasts, keratinocytes, inammatory cells, ECM,
growth factors, cytokines, bacteria, and the microcirculation. An interesting aspect of the venous ulcer milieu is the
presence of chronic venous ulcer wound uid. e wound
uid is known to have important properties that aect cellular function, with many of the identied components
being proteases, proteinases, ECM proteins, inhibitors,
MMPs, chemokines, cytokines, and growth factors.69 A signicant number of chemokines (IL-8, MCP-1, MIP-1α, and
RANTES) and cytokines (TNF-α, IL, interferon-γ [IFN-γ],
growth-regulated protein α [GROα], and eotaxin-2) have
been determined to be present in VLU wound uid. Many of
these chemokines and cytokines are produced and secreted
in the ECM and tissue interstitium and in the wound uid
by inammatory cells. ese compounds are important for
the recruitment of leukocytes, the activation of MMPs, tissue destruction, and a persistent inammatory state in the
VLU.69 Because MMPs are involved in the pathogenesis of
VLUs, it is essential to examine data that have evaluated
proteinases in the VLU wound uid. e collagenase activity of venous ulcer uid is 116-fold more than that found
in normal acute wound uid. e collagenase activity
decreases in VLUs, demonstrating healing at 2 weeks.
e venous ulcer uid causes inhibition of broblast proliferation and induces changes that are consistent with cellular
senescence.
48,72
It also inhibits the growth of neonatal broblasts, causing the majority of cells to remain in the G1 or
G2 phases of the cell cycle (i.e., unable to enter the S phase,
thereby blocking DNA synthesis). When compared with
broblasts treated with bovine serum albumin, the wound
uid demonstrated dose-dependent inhibition at a concentration of 500 mg/plate and was not toxic (by trypan blue
exclusion assay). e normal proliferation of neonatal broblasts treated with VLU wound uid can be reversed by heat
inactivation of the wound uid or by the removal and placement of the cells in 10% serum.
73
In addition to broblasts,
VLU wound uid inhibits the proliferation of endothelial
cells and keratinocytes in part by inhibiting DNA synthesis. Although the specic inhibitory components in VLU
wound uid are not known, there is evidence to suggest
that this active inhibitory substance resides in the less than
30 kDa fraction, and has two to three times the inhibitory
eect on cells than the fraction that is greater than 30 kDa.74
e inhibitory eect of wound uid can be reversed by heating to 100°C, and at concentrations of 2% and 4%, wound
uid causes cell death. Venous ulcer uid was demonstrated
to inhibit the expression of MAPK, specically ERK1 and
ERK2, with a simultaneous decrease in the proliferation
70,71
of neonatal broblasts.62 e mechanism of cell inhibition
by wound uid involves, in part, downregulation of the
phosphorylated pRb tumor suppression gene and cyclin D1
via inhibition of the Ras-dependent MAPK pathway.75 e
compound(s) in VLU uid that cause(s) changes in cellular
function will be a focus of future investigations. Identifying
the inhibitory substances in wound uid will be important
for understanding its molecular eects on cell behavior,
regulation, transcriptional, and pre- and post-translational
alternations, as well as phenotypic alterations, and will
advance our knowledge for the better treatment of venous
ulcers. Biomarkers found in VLU uid may be useful for
determining healing potential, as well as therapeutic targets
in the treatment of VLUs.
69
7.8.2 The ECM and MMPs
e ECM is an important structural and functional scaffolding that is made up of proteins that are necessary for
cell function, wound repair, epithelialization, blood vessel
support, cell dierentiation and signaling, and cellular
migration. e ECM is composed of many proteins and
glycoproteins, including collagen, elastin, bronectin,
vitronectin, aggrecan, entactin, proteoglycans, glycosaminoglycans, growth factors, integrins, tenascin, brin,
and laminin.
providing a substrate in which keratinocytes can migrate
in order to ultimately establish skin coverage in both acute
and chronic wounds.67 Abnormal ECM metabolism in
wounds has been an area of interest and investigation. e
MMPs are proteases that are involved in both healthy and
disease states involving ECM turnover. MMPs are highly
homologous, zinc-dependent endopeptidases that belong
to a large group of proteases called the metzincins, and
they are able to cleave most of the constituents of the ECM.
At least 26 identied and characterized MMPs are classied according to their substrate specicity and structural
similarities. eir four major subgroups are interstitial
collagenases, gelatinases, stromelysins, and membranetype MMPs. Other MMPs are in dierent subgroups, such
as the matrilysins.
tors of MMPs are the tissue inhibitors of MMP (TIMPs).
Molecules such as trocade (Ro 32-3555), marimastat
(BB-25160 and BB-94), and Ro 28-2653 are also known
to inhibit MMPs, and are useful in studying the kinetics
and mechanisms of MMPs in biological systems.79 In an
early report evaluating VLU wound uid compared with
uid from acute wounds, it was found that the chronic
wound uid contained up to a 10-fold increase in the levels of MMP-2 and MMP-9 (gelatinases), as well as showing
increased activity of these enzymes, suggesting a high tissue turnover.80 Increased levels of MMP-1 and gelatinase
activity from the exudates of chronic VLUs have been conrmed by other investigators, and doxycycline inhibition
studies suggested that the protease activity was such that
the cell source was from broblasts, mononuclear cells,
keratinocytes, or endothelial cells, but not neutrophils.
69,76,77
e ECM is particularly important for
78,79
e naturally occurring inhibi-
81
82 Venous ulcer formation and healing atcellularlevels
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It was essential to determine the source of collagenase
activity, since bacteria also produce collagenase and are
abundant in venous ulcers. An important distinguishing
feature of human collagenase is that it degrades collagen
in specic 3/4 and 1/4 fragments, whereas bacterial collagenase degrades collagen randomly in a non-specic manner. It was determined that the collagenase from VLU uid
degraded collagen in the specic 3/4 and 1/4 fragments
that are indicative of human collagenase.81 e changes
noted in the MMP levels and activity in venous ulcers are
not specic to just venous disease, and similar alterations
are found in other inammatory wounds, including burns
and pressure ulcers.82 Other investigators have also demonstrated increased levels of MMP-1 and decreased levels
of TIMP-1 in VLU uid. Importantly, VLU uid leads to
the signicant overexpression of MMP-1 and MMP-3 in
newborn broblasts compared to broblasts treated with
acute wound uid or fetal calf serum.83 In another interesting study, VLU uid was compared to acute wound uid
and assessed for MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). NGAL binds covalently to MMP9,
inhibiting the deactivation of MMP-9 and increasing its
activity. As expected, MMP-9 and NGAL were signicantly elevated in VLU uid compared to controls. elevels of MMP-9 and NGAL in VLU uid decreased at 4 and
8 weeks in VLUs that healed.84 e production of TIMPs
can have a signicant inuence on MMP expression. In
an in vitro study, broblasts cultured from venous ulcers
demonstrated a marked reduction in MMP-1 and MMP-2
level and activity and a signicant increase in TIMP-1 and
TIMP-2 production. e authors concluded that the inhibition of broblast proteinase activity by TIMPs causes
impaired reorganization of the ECM in chronic wounds,
leading to delays in healing.85 is study indicated that
although there is elevated proteinase activity in the venous
ulcer wound and wound uid, cellular components studied in vitro (in this case, broblasts) compensate by altering their expression of MMPs and TIMPs.
e abnormalities in the structure and the healing process seen in lipodermatosclerotic skin have also been attributed to the MMP pathway. In one study, dermal biopsies
were obtained from lipodermatosclerotic skin, compared
with healthy skin, and analyzed by immunohistochemistry,
reverse transcriptase polymerase chain reaction, immunoblot, and zymography analysis. e study found that lipodermatosclerotic skin had increased expression of mRNA and
protein for MMP-1, MMP-2, and TIMP-1, and increased
levels of active MMP-2. In addition, there was an increase in
the proMMP-1–TIMP1 complex, indicating that the overexpression of proteinase was bound to TIMP.
86
As assessed
by immunohistochemistry, both MMP-1 and MMP-2
were predominantly localized in the basal and suprabasal
layers of the epidermis, the perivascular region, and the
reticular dermis, and signicantly reduced expression of
TIMP-2 was found in the basement membrane of the dis-
86
eased skin.
is demonstrates that in lipodermatosclerotic
skin—a pre cursor to venous ulcer formation—excessive
and unrestrained MMP activity and ECM turnover occurs,
especially in the areas of the dermis, the epidermis, and the
perivascular region. A consistent nding is the presence
of MMPs in the perivascular region (see below in Section
7.7.3). A consideration is that MMPs may cause abnormalities in tissue perfusion, or aect angiogenesis and the
microvasculature. In an elegant study, investigators compared VLU uid with control acute wound uid (donor skin
gra sites) and tested both uids in an in vitro angiogenesis
model by measuring tubule length. e venous ulcer uid
caused a signicant reduction in the formation of tubules
and their length (490 ± 130 mm) compared with the control
uid (1740 ± 320 mm, P < 0.05). When a synthetic inhibitor
of MMP-2 and MMP-9 was added to chronic venous ulcer
uid, angiogenesis increased signicantly (870 ± 220 mm,
P < 0.05).87 e proteolytic activity of MMP-9 can generate
angiostatin from plasminogen, which inhibits the proliferation of human microvascular endothelial cells. Endostatin
is also antiangiogenic and can be activated by MMPs. ese
data raise the possibility that MMPs in venous ulcer wound
uid may have signicant antiangiogenic eects and may
disrupt the microcirculation in the perivascular regions,
thereby inhibiting wound healing.
7.8.3 Modulation and activation of MMPs
MMPs are synthesized in a pro-enzyme form. e proenzymes have a cysteine domain called the cysteine switch
that interacts with the zinc active binding site, preventing
activation and substrate degradation. e cysteine switch
is cleaved prior to the pro-enzyme becoming active.78 e
excess proteolyt ic activity i n VLUs has been found to degrade
essential plasminogen, activating proMMP to MMP, which
is necessary for brinolysis and cell migration. MMPs also
inhibit plasmin production by keratinocytes, which may
lead to reduced cell migration.88 Important to the healing wound is FXIII, which impacts collagen cross-linking.
FXIII has the ability to modulate the detrimental eects of
MMPs. In an in vitro study, the investigators evaluated the
eects of increasing concentrations of collagenase and FXIII
on broblast survival, as assayed by the MTT colorimetric
test. At high concentrations of collagenase (2 mg/mL), 95%
of broblasts were killed, and FXIII (5 U/mL) was unable to
mitigate the eect. However, at lower collagenase concentrations (0.5–1 mg/mL), the addition of FXIII was able to
abrogate the eects of collagenase and increase broblast
survival. ese data were consistent with clinical ndings that the topical application of FXIII has the ability to
improve venous ulcer healing.
overload has been found in the serum and dermis of the
limbs of patients with venous ulcer, compared with control
subjects. A concomitant elevation in MMP-9 activity was
also present in patients with VLUs. e importance of iron
overload in venous ulcer tissue is that it can cause oxidative
stress and the production of free radicals or reactive oxygen
19
In addition to FXIII, iron
7.8 Wound fluid environment andMMPs 83
Cells: Mc
Cells: Et, Kt
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species. e authors suggest that elevated iron deposits in
the limbs are released into the serum with the activation of
MMPs and reactive oxygen species, impairing ulcer healing.89 Other investigators have also found increased ferritin
and overall oxidative stress as measured by 8-isoprostane
and total antioxidant status in VLU uid. Importantly, the
levels of ferritin and oxidative stress were signicantly lower
in patients with healing VLUs versus those that had nonhealing VLUs.
90
As mentioned previously, plasminogen is essential in
MMP regulation.88 Urokinase-type plasminogen activator (uPA) functions as a brin-independent plasminogen
activator in a cell-bound fashion, and when uPA is bound
to its receptor uPAR, the activity of uPA is potentiated.
Comparing venous ulcers with normal dermis, one study
found that both the transcriptional products and the proteins of uPA and uPAR were overexpressed in venous ulcers.
Localization of uPA and uPAR by immunohistochemistry
determined that these proteins were present in the dermis
and in the pericapillary regions.91 One could hypothesize
that uPA is crucial for maintaining proteolytic activity and
likely has a role in the activation of MMPs via plasmin in
the pathogenesis of venous ulcers.
Two important molecules in the activation of MMPs
are MT1-MMP and the extracellular MMP inducer
(E MMPR IN; C D147).
78,92
Utilizing an immunohistochemistry assay, MMP-2, MT1-MMP, MT2-MMP, and EMMPRIN
were found to be signicantly elevated in the venous ulcer
dermis, and only EMMPRIN and MMP-2 were overexpressed in the perivascular regions in venous ulcer biopsies. ese data indicate the presence of MMP activators
in venous ulcer tissue that favor extracellular turnover
and unrestrained MMP activation.92 In another study
evaluating healing versus non-healing VLUs, the investigators determined that in healing ulcer tissue there were
increased levels of PDGF-AA, but no dierence in MMP or
EMMPRIN levels. In the same study, the venous ulcer uid
of healing ulcers versus non-healing ulcers demonstrated
elevated levels of PDGF-AA and TIMP-2 and low levels of
MMP-2. ese ndings are signicant as they help to dene
the factors that are important for ulcer healing and support
the theory that elevated proteinase activity (MMP-2 and
MMP-9) favors a non-healing environment. In addition, the
growth factor PDGF-AA appears to be essential to promoting healing.93 e activation of MMPs and their potential
involvement in ulcer formation is summarized in Figure 7.4.
Cells: Kt, Fb, Et
EMMPRIN
MMP transcription
translation
pro-MMP-1, -2, -9
pro-MT1–MMP
pro-MT2–MMP
MT–MMP
Pro-MMPs
Plasminogen
uPA
TGF-β1
pro-uPA
Figure 7.4 Matrix metalloproteinase activation and unbalanced proteinase activity leading to venous ulcer formation.
Schematic diagram of the activation of MMPs. EMMPRIN activation leads to the synthesis of proMMPs. In addition, iron
overload and reactive oxygen species lead to the expression of MMPs. Furthermore, pro-uPA is synthesized and converted to uPA by TGF and binds to its receptor, uPAR, which potentiates the conversion of plasminogen to plasmin. The
proMMPs are secreted in their inactive forms and are activated by both plasmin and membrane-type MMPs (MT1-MMP
and MT2-MMP). Active MMPs in the wound fluid cause tissue degradation, anti-angiogenesis, and fibroblast and keratinocyte inhibition, promoting non-healing venous ulcers (negative factors). Factors promoting the healing of venous ulcers
are the presence of tissue inhibitors of MMP (TIMP), growth factors such as platelet-derived growth factor AA (PDGF-AA),
and factor XIII (FXIII) (positive factors). Cells involved are keratinocytes (Kt), fibroblasts (Fb), endothelial (Et) cells, macrophages (Mc), and leukocytes.
Plasmin
uPA
uPAR
Fe, ROS
Active MMPs
Wound fluid
Active MMPs
Antiangiogenesis
+ Factors
Growth factors
PDGF AA
TIMPs
Ulcer
– Factors
Hypoxia
FXIII
84 Venous ulcer formation and healing atcellularlevels
https://t.me/med1917
7.8.4 Regulation of MMPs
e regulation of MMP production in venous ulcers and
lipodermatosclerotic tissue is complex. Post-translational
modications of MMPs are essential for activity, and are
likely regulated by TGF-β1. Dermal broblasts and leukocytes are major sources of MMPs, especially MMP-
2.94 e interplay of MAPK and MMP activation has also
been investigated in broblasts. e cytokine TNF-α has
been demonstrated to induce MMP-19 expression, which
is inhibited by blocking the MAPK pathways ERK1 and
ERK2 with PD98059 and p38 with SB203580. In addition,
adenovirus-mediated induction of ERK1 and ERK2 in combination with p38 resulted in potent MMP-19 expression in
broblasts, and the activation of c-JNK also produced abundant proMMP-19.95 ese data, as well as ndings of MAPK
alterations in venous ulcer broblasts due to the eects of
wound uid,
62,63,75
indicate the important regulatory functions of MAPK and proteolytic activity in dermal broblasts and their implications in venous ulcer pathogenesis.
95
7.9 IMPORTANT MARKERS FOR
VLUHEALING
VLU healing reaches between 60% and 70% at 12–24 weeks,
and the principle treatment applied is compression.
is essential to understand the pathophysiology of VLUs so
that biomarkers that are predictive of VLU healing and
potential therapeutic targets can be developed.69 Several
works have already indicated that VLU healing is associated with a decrease in MMP-9 and NGAL,84 as well as
a reduction in oxidative stress.90 In a study of 40 patients
with healing versus non-healing VLUs of greater than 8
weeks’ duration, patients underwent tissue biopsy at the
VLU edge and wound uid evaluation at the initial visit.
Evaluation of VLU healing occurred aer 8 weeks. In healing VLUs, there were signicantly higher (P < 0. 0 01) leve l s
of PDGF-AA in the perivascular region, and in the wound
uid there were signicantly increased levels of PDGF-AA
and a decreased ratio of MMP-2:TIMP-2 (P = 0.0001).93
Collagen turnover and remodeling is an important function of healing VLUs. In a study evaluating VLU biopsies in
healed patients (n = 12) and non-healed patients (n = 15), a s
well as controls (n = 15), aer applying compression bandaging for 12 months, the degradation products of collagen and collagen turnover were determined. Healed VLUs
had signicantly (P < 0.001) elevated levels of degraded
collagen and type III collagen (P = 0.005, as measured by
collagen III N-terminal propeptide), and elevated levels of
MMP-1 (P < 0.001; MMP-1 is important in tissue remod-
98
eling during healing).
e role of TGF-β1 in VLU heal-
ing was investigated in a study of 80 patients treated with
multilayer compression bandaging. In the wound uid and
serum, cytokines and factors reecting the processes of
inammation (IL-1 and TNF-α), proteolysis (proMMP-2
and proMMP-9), angiogenesis (bFGF and VEGF), and
96,97
It
matrix deposition/ proliferation/brosis (TGF-β1) were
measured. Interestingly, ulcer healing at 5 weeks only
correlated signicantly with increased concentrations of
TGF-β1 in the VLU uid.99 An elegant analysis of cytokine
levels and venous ulcer healing determined that untreated
ulcers typically display high levels of pro- inammatory
cytokines, including several interleukins, TNF-α, and
IFN-γ. Aer 4 weeks of compression therapy, the levels of
pro-inammatory cytokines decreased signicantly and
the wounds began to heal. e levels of TGF-β1 increased
signicantly as the ulcers improved. When specic cytokine levels were related to the percentage of healing, it was
found that those with higher levels of pro-inammatory
cytokines, including IL-1 and IFN-γ, healed signicantly
better than those with lower levels prior to compression.
Treatment with compression therapy resulted in healing
that was coupled with reduced pro-inammatory cytokine
levels and higher levels of the anti-inammatory cytokine
IL-1 receptor antagonist.
100
In another study evaluating
patients with VLUs, tissue biopsies were obtained at the
initial visit and aer 4 weeks of compression therapy. At
4 weeks, signicant decreases in both mRNA and protein
were seen for MMP-3 (stromelysin-1) and MMP-9 (gelatinase-B). In addition, in those patients who had greater than
40% healing of the VLU versus those that had less than
40% healing, signicant decreases in MMP-1, MMP-2, and
MMP-3 were identied.
101
ese studies indicate the com-
plex interplay of collagen turnover, MMPs, pro-inammatory and anti-inammatory cytokines, and TGF-β1. e
importance of balanced and temporal MMP and cytokine
function, and the key role of TGF-β1 in promoting VLU
healing, were also demonstrated.
7.10 CONCLUSION
VLU pathophysiology is a complex process that involves
the many changes discussed in this chapter, including
genetic and environmental inuences, alterations in shear
stress and injury to the glycocalyx with endothelial activation, the inammatory response due to leukocytes acting
on the venous endothelium and microcirculation, alterations in cellular functions, with dysregulation of important cellular elements (broblasts and keratinocytes), the
overexpression of chemokines, cytokines, dysregulation of
signaling pathways such as TGF-β and MAPK, and MMPs
and their impact on the ECM. Another component that
perpetuates an inammatory and non-healing state is the
inhibitory environment of VLU uid, causing a signicant negative inuence on cellular growth and healing, in
addition to some regulatory pathways. From this review
and the research examined, several observations and conclusions can be summarized, as listed in the Guidelines.
Venous ulcer pathophysiology involves systemic and local
processes. It is likely that targeting only one system may
not cause a clinical change in ulcer healing. It is likely that
several systems need to be intervened in so as to achieve
References 85
https://t.me/med1917
clinical response and decrease recurrence. Our current
understanding of VLU development is just the tip of an
iceberg. However, as monumental as it may seem, the task
of acquiring knowledge through careful scientic investigation must progress. As specialists in venous diseases, we
must better understand the complexities of venous ulcer
Guidelines 1.6.0 of the American Venous Forum on venous ulcer formation and healing at cellular levels
No. Guideline
1.6.1 We recommend a basic practical knowledge of venous physiology and
venouslegulcer pathophysiology for all practitioners caring for venous legulcers.
1.6.2 Age, genetic, and environmental factors predispose to venous ulcers. B
1.6.3 Shear stress, glycocalyx injury, and expression of adhesions molecules with venous
endothelial activation allow attachment of leukocytes and are key steps in the
progression of chronic venous insufficiency.
1.6.4 Leukocyte activity and interaction with endothelial cells initiate a cascade of
inflammatory events.
1.6.5 Macrophages play a major role in ulcer formation. C
1.6.6 Dysfunctional leukocytes, senescent fibroblasts, and keratinocytes contribute to
delayed ulcer healing.
1.6.7 Key regulatory cell cycle proteins (p21 and pRb) affect fibroblast proliferation and
delay wound healing.
1.6.8 Venous ulcer fluid has elevated inhibitory cytokines and matrix metalloproteinases
(MMPs). MMPs play an integral role in venous ulcer formation.
1.6.9 Factor XIII, plasminogen, and extracellular MMP inducer (EMMPRIN) modulate
MMP activity and contribute to venous ulcers.
pathology and focus our resources on several issues, such as
the regulation of shear stress and the glycocalyx, leukocytes
in the microcirculation, the regulation of the cells involved
in healing, wound uid and its eect on the ulcer environment, and the eects and regulation of chemokines, cytokines, and MMPs.
Grade of evidence
(A:highquality; B:
moderate quality; C: low or
very low quality)
Best practice
B
B
B
B
A
C
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