Role of angiogenesis and angiogenic factors in acute and chronic wound healing

Abstract

Angiogenesis plays a crucial role in wound healing by forming new blood vessels from preexisting vessels by invading the wound clot and organizing in to a microvascular network throughout the granulation tissue. This dynamic process is highly regulated by signals from both serum and the surrounding extracellular matrix (ECM) environment. Vascular endothelial growth factor, angiopoietin, fibroblast growth factor and transforming growth factor beta are amongst the potent angiogenic cytokines in wound angiogenesis. Specific endothelial cell ECM receptors are critical for morphogenetic changes in blood vessels during wound repair. In particular integrin (αvβ3) receptors for fibrin and fibronectin, appear to be required for wound angiogenesis: αvβ3 is focally expressed at the tips of angiogenic capillary sprouts invading the wound clot, and any functional inhibitors of αvβ3 such as monoclonal antibodies, cyclic RGD peptide antagonists and peptidomimetics rapidly inhibit granulation tissue formation. Inspite of clear knowledge about influence of many angiogenic factors on wound healing, little progress has been made in defining the source of these factors, the regulatory events involved in wound angiogenesis and in the clinical use of angiogenic stimulants to promote repair.

Full text

Topic: Current Concepts in Wound Healing
© 2015 Plastic and Aesthetic Research | Published by Wolters Kluwer - Medknow 243
INTRODUCTION
Neovascularization or angiogenesis is important for
wound healing as it involves the growth of new capillaries
to form granulation tissue.[1‑4] Three to five days after
tissue injury, new capillaries become visible in the wound
bed as granulation tissue, which acts as a matrix for
proliferating blood vessels, migrating fibroblasts and new
collagen.[5] Impaired granulation is a hallmark of chronic
wounds encountered with diabetes and venous or arterial
insufficiency.
In 1960s, research began in the field of angiogenesis
to determine how new blood vessels enhance solid
tumor growth.[6] Physiologists later discovered that
neovascularization occurs during tissue regeneration.[7]
Proliferating capillaries bring oxygen and micronutrients
to growing tissues and remove catabolic waste products.
These vessels are present in the endothelium that secretes
paracrine factors to promote survival of adjacent cells by
preventing apoptosis or programmed cell death.[8] Because
Role of angiogenesis and angiogenic
factors in acute and chronic wound healing
Thittamaranahalli Muguregowda Honnegowda1, Pramod Kumar1,2,
Echalasara Govindarama Padmanabha Udupa3, Sudesh Kumar4, Udaya Kumar4, Pragna Rao3
1Department of Plastic Surgery and Burns, Kasturba Medical College, Manipal 576104, Karnataka, India.
2Department of Plastic Surgery, King Abdulaziz Specialist Hospital, Sakaka 42421, Al‑Jouf, Saudi Arabia.
3Department of Biochemistry, Kasturba Medical College, Manipal 576104, Karnataka, India.
4Department of Surgery, District Government Hospital, Udupi 576108, Karnataka, India.
Address for correspondence: Dr. Pramod Kumar, Department of Plastic Surgery and Burns, King Abdulaziz Specialist Hospital,
Sakaka 42421, Al‑Jouf, Saudi Arabia. E‑mail: pkumar86@hotmail.com
ABSTRACT
Angiogenesis plays a crucial role in wound healing by forming new blood vessels from preexisting vessels
by invading the wound clot and organizing into a microvascular network throughout the granulation
tissue. This dynamic process is highly regulated by signals from both serum and the surrounding
extracellular matrix environment. Vascular endothelial growth factor, angiopoietin, broblast growth
factor and transforming growth factor‑beta are among the potent angiogenic cytokines in wound
angiogenesis. Specic endothelial cell ECM receptors are critical for morphogenetic changes in blood
vessels during wound repair. In particular integrin (αvβ3) receptors for brin and bronectin, appear to
be required for wound angiogenesis: αvβ3 is focally expressed at the tips of angiogenic capillary sprouts
invading the wound clot, and any functional inhibitors of αvβ3 such as monoclonal antibodies, cyclic
RGD peptide antagonists, and peptidomimetics rapidly inhibit granulation tissue formation. In spite of
clear knowledge about inuence of many angiogenic factors on wound healing, little progress has been
made in dening the source of these factors, the regulatory events involved in wound angiogenesis and
in the clinical use of angiogenic stimulants to promote repair.
Key words:
Angiogenic factors, endothelium, extracellular matrix protein, granulation tissue, wound healing
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How to cite this article: Honnegowda TM, Kumar P, Udupa EG,
Kumar S, Kumar U, Rao P. Role of angiogenesis and angiogenic factors
in acute and chronic wound healing. Plast Aesthet Res 2015;2:243-9.
Received: 20-10-2014; Accepted: 28-01-2015

Plast Aesthet Res || Vol 2 || Issue 5 || Sep 15, 2015244
angiogenesis is required for wound healing, its induction
is beneficial in many clinical situations for achieving
wound closure.
PHYSIOLOGICAL CONTROL OF
ANGIOGENESIS
Angiogenesis plays a critical role in wound healing. By
developing capillary sprouts, which digest endothelial
cells and invade the extracellular matrix (ECM) stroma
after penetrating through the underlying vascular
basement membrane (VBM), and form tube‑like structures
that continue to extend, branch, and form networks.
During angiogenesis capillary advancement in ECM occurs
by endothelial cell proliferation and direction of growth
is guided by chemotaxis from the target region. The
interaction among endothelial cells, angiogenesis factors
and surrounding ECM proteins is temporally and spatially
synchronized.[9,10]
Angiogenesis can be induced in response to injury via
pro‑ and anti‑angiogenic factors present throughout the
body. Pro‑angiogenic factors consist of thrombin, fibrinogen
fragments, thymosin‑β4 and growth factors. Angiogenic
growth factors are stored in platelets and inflammatory
cells that circulate in the bloodstream, and are sequestered
within the ECM. The production of these factors is
regulated by genes expressed in response to hypoxia and
inflammation, such as hypoxia‑inducible factors (HIF) and
cyclooxygenase‑2 (COX‑2).[11‑13] In contrast, angiogenesis
inhibitor factors suppress blood vessel growth.[14,15] Some
inhibitors circulate in the blood stream at low physiological
levels while others are stored in the ECM surrounding
blood vessels. Vascular growth is suppressed when
there is a physiological balance between angiogenesis
stimulators and inhibitors.[15] Immediately following injury,
however, angiogenic stimuli are released into the wound
bed, and a shift occurs in regulators favoring vascular
growth [Figure 1].
THE ANGIOGENESIS CASCADE
Angiogenesis occurs as an orderly cascade of molecular
and cellular events in the wound bed:
1. Endothelial cell surface has receptors to which angiogenic
growth factors bind in preexisting venules (parent vessels);
2. Growth factor‑receptor binding activates signaling
pathways within endothelial cells;
3. Proteolytic enzymes released by activated endothelial cells
dissolve the basement membrane of surrounding parent
vessels;
4. Endothelial cells proliferate and sprout outward through
the basement membrane;
5. Endothelial cells migrate into the wound bed using
integrins (αvβ3, αvβ5 and αvβ1) which are cell surface
adhesion molecules;
6. Matrix metalloproteinases (MMPs) dissolve the surrounding
tissue matrix in the path of sprouting vessels;
7. Vascular sprouts form tubular channels that connect to
form vascular loops;
8. Vascular loops differentiate into afferent (arterial) and
efferent (venous) limbs;
9. New blood vessels mature by recruiting mural cells
(smooth muscle cells and pericytes) to stabilize the
vascular architecture;
10. Blood flow begins in the mature stable vessel.
These complex growth factor‑receptor, cell‑cell and cell‑matrix
interactions characterize the angiogenesis process, regardless
of the stimuli or its location in the body.
THE ANGIOGENESIS MODEL OF
WOUND HEALING
Wound healing occurs in four major overlapping stages:
(1) hemostatic, (2) inflammatory stage, (3) proliferative
stage, and (4) remodeling stage. Although granulation
is assigned to the proliferative stage, angiogenesis is
initiated immediately after tissue injury and is mediated
throughout the wound healing process.
Step 1: Angiogenesis initiation
Basic fibroblast growth factor (bFGF) stored within intact cells
and the ECM is released from damaged tissue.[16] Bleeding and
hemostasis in a wound also initiate angiogenesis. Cellular
receptors for vascular endothelial growth factor (VEGF) are
upregulated by thrombin in the wound.[17] Endothelial cells
exposed to thrombin also release gelatinase A (MMP‑2), which
promotes the local dissolution of basement membrane, a
necessary early step of angiogenesis.[18] Platelets release
multiple growth factors, including platelet‑derived
growth factor (PDGF), VEGF, transforming growth
factor (TGF‑α, TGF‑β), bFGF, platelet‑derived endothelial
cell growth factor and angiopoietin‑1 (Ang‑1). These factors
stimulate endothelial proliferation, migration and tube
formation.[19‑22]
Step 2: Angiogenesis amplication
Macrophages and monocytes release numerous angiogenic
factors, including PDGF, VEGF, Ang‑1, TGF‑α, bFGF,
interleukin‑8 (IL‑8) and tumor necrosis factor alpha into
the wound bed during the inflammatory phase amplifying
angiogenesis further.[23,24] Several growth factors (PDGF, VEGF
and bFGF) synergize in their ability to vascularize tissues.[25]
Proteases that break down damaged tissue matrix further
release matrix‑bound angiogenic stimulators. Enzymatic
cleavage of fibrin yields fibrin fragment E, which
stimulates angiogenesis directly and also enhances the
Figure 1: Angiogenesis is a balance between stimulators (growth factors)
and inhibitors as shown in this model

Plast Aesthet Res || Vol 2 || Issue 5 || Sep 15, 2015 245
effects of VEGF and bFGF.[26] Expression of the inducible
COX‑2 enzyme during the inflammatory stage of healing
also leads to VEGF production and other promoters of
angiogenesis.[27]
Step 3: Vascular proliferation
Hypoxia is an important driving force for wound
angiogenesis. Expression of gene HIF‑1α, due to hypoxic
gradient between injured and healthy tissue triggers
VEGF production.[24,28] VEGF is present in both wound
tissue and exudate.[28,29] VEGF is also known as vascular
permeability factor since it increases permeability of
capillaries.[30] Hypoxia also leads to endothelial cell
production of nitric oxide (NO). NO promotes vasodilation
and angiogenesis to improve local blood flow.[31]
Step 4: Vascular stabilization
Vascular stabilization is governed by Ang‑1, tyrosine kinase
with immunoglobulin‑like and EGF‑like domains 2 (Tie‑2),
smooth muscle cells and pericytes. Production of PDGF
and recruitment of smooth muscle cells and pericytes to
the newly forming vasculature are regulated by binding
of Ang‑1 to its receptor Tie‑2 on activated endothelial
cells.[32‑34] A PDGF deficiency leads to poorly‑formed
immature blood vessels.[35]
Step 5: Angiogenesis suppression
Angiogenesis is suppressed at the terminal stages of
healing.[36] As tissue hypoxia is restored, and inflammation
subsides, the level of growth factors decline in the wound.
Pericytes which stabilize endothelial cells secrete an
inhibitory form of activated TGF‑β that impedes vascular
proliferation.[34,37,38] A cleavage product of collagen XVIII,
endostatin, is present surrounding the VBM, and it inhibits
wound vascularity.[39,40]
WOUND ANGIOGENIC STIMULATORS
AND INHIBITORS
A number of angiogenic stimulators have been identified
in wound sand others are likely to exist that play an
important role in the repair [Table 1]. The stimulators
in wound fluids are growth factors known to increase
endothelial cell migration and proliferation in vitro.[41]
The FGF comprises of 23 homologous structures that
are small polypeptides with a central core containing
140 amino acids. Acidic FGF and bFGF are the first few
to be discovered and are now designated as FGF‑1 and
FGF‑2, respectively.[42] Both are preferentially involved
in the process of angiogenesis.[43,44] These compounds
are polypeptides of about 18 kDa, single chained and
nonglycosylated. They transmit their signals through
FGF receptor‑4 (FGFR‑4) high‑affinity, protein family of
transmembrane tyrosine kinases (FGFR‑1 to FGFR‑4), that
bind to different FGFs with different affinities. The strong
interactions of FGF‑1 and FGF‑2 with glycosaminoglycans,
such as heparin sulfate present in the ECM,[45] makes the
FGFs stable against thermal, proteolytic denaturation and
limits its diffusibility. Thus, the ECM acts as a reservoir for
pro‑angiogenic factors. Most members of the FGF family
act as a broad spectrum mitogen that stimulates the
proliferation of mesenchymal cells of mesodermal origin,
as well as ectodermal and endodermal cells.
FGF‑1 and FGF‑2 are synthesized by a variety of cell types
including inflammatory cells and dermal fibroblasts that
are involved in angiogenesis and wound healing. When
liberated from ECM, they act on the endothelial cells
in a paracrine manner, or when released by endothelial
cell they act in an autocrine manner promoting cell
proliferation and differentiation. During the formation of
granulation tissue, FGF‑2 promotes cell migration through
surface receptors for integrins, which mediate the binding
of endothelial cells to ECM.[44]
Vascular endothelial growth factor increase vaso‑permeability
by increasing the fenestration and hydraulic conductivity.
This allows leakage of fibrinogen and fibronectin,
which are essential for the formation of the provisional
ECM.[46‑48] The ECM is produced in large quantities by
the epidermis during wound healing.[49] Low oxygen
tension that occurs in tissue hypoxia is a major inducer
of VEGF[50] and its receptors.[51] Thus, cell disruption and
hypoxia appear to be strong initial inducers of potent
angiogenesis factors at the wound site. VEGF family
currently includes VEGF‑A, VEGF‑B, VEGF‑C, VEGF‑D,
VEGF‑E and placental growth factor.[52] VEGF‑A is a
homodimer glycoprotein whose subunits are linked by
2 disulfide bonds. VEGF‑A is synthesized from internal
rearrangements (“alternative splicing”) of mRNA. Thus,
there is the production of 7 isoforms with 121 to 206
amino acids.[53‑55] Among these, the VEGF121, VEGF165,
VEGF189 and VEGF206 are the predominant isoforms.[56]
These isoforms show similar biological activities, but differ
in their binding properties to heparin and ECM.[57]
Vascular endothelial growth factor is a potent vascular
endothelial cell‑specific mitogen that stimulates endothelial
cell proliferation, microvascular permeability and regulates
of several endothelial integrin receptors during sprouting
of new blood vessels.[58] Furthermore, VEGF also acts
as a survival factor for endothelial cells by inducing the
expression of an anti‑apoptotic protein B‑cell lymphoma 2.[59]
TGF‑β stimulates the formation of granulation tissue by
acting as a chemoattractant for neutrophils, macrophages
and fibroblasts. Hence, TGF‑β is an important modulator
of angiogenesis during wound healing by regulating cell
Table 1: Angiogenic stimulators and inhibitors
Stimulators Inhibitors
aFGF (FGF-1) Thrombospondin-1
bFGF (FGF-2) Tissue inhibitors of matrix metalloproteinases
TGF‑α Interferon alpha/beta/gamma
TGF‑β Angiostatin
PGE2 Endostatin
TNF‑α
VEGF
EGF
FGF: Fibroblast growth factor, aFGF: Acidic broblast growth factor,
bFGF: Basic broblast growth factor, TGF‑α: Transforming growth factor‑alpha,
TGF‑β: Transforming growth factor‑beta, VEGF: Vascular endothelial growth
factor, EGF: Endothelial growth factor, PGE2: Prostaglandin E2

Plast Aesthet Res || Vol 2 || Issue 5 || Sep 15, 2015246
proliferation, migration, capillary tube formation and
deposition of ECM.[60,61]
The angiopoietins are members of the VEGF family, which
is largely specific for vascular endothelium. They include a
naturally occurring agonist, Ang‑1, and antagonist, Ang‑2,
both of which act by means of the Tie‑2 receptor. Two
new angiopoietins, Ang‑3 in mice and Ang‑4 in humans,
have been identified, but their function in angiogenesis is
unknown.[62]
Mast cell tryptase, stored in granules of activated mast cells,
is an additional angiogenesis factor that directly degrades
the ECM components or release matrix‑bound growth
factors by its proteolytic activity,[63,64] and acts indirectly
by activating latent matrix metalloproteases. The addition
of tryptase to microvascular endothelial cells cultured on
a basement membrane matrix (matrigel) caused a marked
increase in capillary growth. Furthermore, tryptase can
induce endothelial cell proliferation in a dose‑dependent
manner, whereas specific tryptase inhibitors suppress the
capillary growth.[65]
IMPAIRED ANGIOGENESIS IN CHRONIC
WOUNDS
Angiogenesis is impaired in all chronic wounds leading
to further tissue damage results from chronic hypoxia
and impaired micronutrient delivery. Specific defects have
been identified in diabetic ulcers, venous insufficiency
ulcers and ischemic ulcers.
Diabetic ulcers
Patients with diabetes show abnormal angiogenesis in
various organs. Vasculopathies associated with diabetes
include abnormal blood vessel formation (e.g. retinopathy,
nephropathy) and accelerated atherosclerosis leading
to coronary artery disease, peripheral vascular disease,
and cerebrovascular disease.[65] However, in diabetics,
angiogenesis is decreased[66] resulting in poor formation
of new blood vessels and thus decreased entry of
inflammatory cells and their growth factors. Growth factors
such as FGF‑2 and PDGF, essential for wound healing
have been found to be reduced in experimental diabetic
wounds models.[67‑70] Furthermore, in rat models, topical
administration of high glucose to wounds was shown to
inhibit the normal angiogenic process,[71] suggesting a direct
role for high glucose levels in diminished angiogenesis.
Vascular endothelial growth factor plays an important
role in vascular growth and has been shown to be
deficient in diabetic wounds in experimental and clinical
models.[72] Studies have shown that modulation of
oxidative damage[73] or inhibition of the receptors for
advanced glycation end products[74] improve wound healing
and were associated with the up‑regulation of endogenous
VEGF. Moreover, VEGF administration improves wound
healing in nondiabetic ischemic wounds[75] and blocking
VEGF with neutralizing antibodies impedes tissue repair.[76]
These studies support the notion that VEGF is critical for
repair in impaired healing states and that the addition
of VEGF could have a potential clinical use.[77] In fact,
Galiano et al.[78] found that topical VEGF accelerates wound
healing in a diabetic mouse model.
Weinheimer‑Haus et al.[79] found that low intensity
vibration (LIV) applied vertically at 45 Hz with peak
acceleration of 0.4 g for 30 min a day for 5 days a week
starting on the day of injury in diabetic mice increases
expression of pro‑healing growth factors and chemokines
(insulin‑like growth factor‑1, VEGF and monocyte
chemotactic protein‑1) in wound environment. Though
there was no evidence of a change in the phenotype
of CD11b+ macrophages, however, LIV resulted in
trend toward a less inflammatory phenotype in the
CD11b2 cells which comprised of fibroblasts, endothelial
cells and/or keratinocytes. These findings indicate that
LIV may exert beneficial effects on wound healing by
enhancing angiogenesis and granulation tissue formation,
and these changes are associated with an increase in
pro‑angiogenic growth factors.[79]
Venous insufciency ulcers
Venous insufficiency ulcers or venous stasis ulcers result
from incompetent valves in lower extremity veins, leading
to venous stasis and hypertension that makes the skin
susceptible to ulceration. Pathological findings associated
with venous stasis ulcers include microangiopathy,
fibrin “cuffing” and trapping of leukocytes within the
microvasculature.[80,81]
Chronic venous stasis ulcer patients have elevated levels
of VEGF in their circulation.[82] This may explain the
vascular permeability and increased transudation of serum
fluid in their wounds. Biopsies of these ulcers reveal
microvessels that are surrounded by fibrin cuffs composed
of fibrin and plasma proteins, such as α‑macroglobulin,
thought to compromise gas exchange.[83‑85] Clinical studies
have shown that transcutaneous oxygen tension may be
up to 85% lower in venous stasis ulcers compared with
normal skin regions.[86] VEGF expression is up‑regulated by
hypoxia, which further exacerbates vascular permeability,
formation of pericapillary fibrin cuffs and compromised
gas exchange, which ultimately reduces growth factor
availability in the wound.[87,88] VEGF promotes the
formation tortuous, aberrant glomeruloid‑like vascular
structures found in granulation tissue.[89] Laboratory
animals treated with VEGF form these glomeruloid
vascular structures within 3 days and are characterized
by poor perfusion.[90] In venous ulcers, the persistence of
glomeruloid vessels may interfere with oxygen delivery
and delay healing. In chronic venous stasis ulcers,
high levels of proteases such as neutrophil elastase,
MMPs and urokinase‑type plasminogen activator are
present.[91] Concomitantly, there are decreased levels
of protease inhibitors, such as plasminogen activator
inhibitor‑2. Excessive protease activity may degrade the
growth factors and destroy granulation tissue.
Ischemic ulcers
Peripheral arterial disease (PAD) may result in severe
ischemia.[92] Reduce tissue perfusion due to ischemia
results in progressive tissue hypoxia, ischemia, necrosis
and skin breakdown. In theory, tissue hypoxia should

Plast Aesthet Res || Vol 2 || Issue 5 || Sep 15, 2015 247
initiate angiogenesis via inducing an HIF‑1α and
angiogenic growth factors. In patients with PAD, serum
levels of hepatocyte growth factor are elevated than in
normal subjects.[93] The tissue compromise caused by
severe macrovascular disease, however, may over dominate
the angiogenic response. Inter‑individual differences in the
ability to mount angiogenesis under hypoxic conditions
also exist among patients with atherosclerosis. Such
variations may explain that patients with PAD are unable
to generate adequate collateral circulation and unable to
heal arterial ulcers despite surgical bypass. Therapeutic
growth factors or other methods designed to stimulate
angiogenesis might benefit patients with a defective
angiogenic capacity. VEGF gene transfer[94] or autologous
transplantation of bone marrow‑derived endothelial
progenitor stem cells[95] improved healing of arterial ulcers
in patients.
ANGIOMODULATORY STRATEGIES
Wound angiogenesis represents a realistic model to study
molecular mechanisms involved in the formation and
remodeling of vascular structures. In particular, the repair
of skin defect offers an ideal model to analyze angiogenesis
as it is easy to control and manipulate this process.[96]
Vessel growth is controlled by the local actions of chemical
mediators, the ECM, metabolic gradients and physical
forces. Manipulation of some of these factors is being tried
to improve healing in experimental wounds.[97] Scientists
are working on mathematical models which describe
the role of angiogenesis as observed during (soft tissue)
wound healing. Through this model manipulation of the
capillary tip, macrophage‑derived chemical attractant profile,
extracellular matrix and fibroblast diffusion coefficient may
be analyzed to enhance wound healing.[98]
CONCLUSION
Angiogenesis is a physiological process that is vital for
normal wound healing. A number of factors regulate
wound angiogenesis, including hypoxia, inflammation
and growth factors. The molecular and cellular events in
angiogenesis have been elucidated, and defects in this
process are present in chronic wounds. Based on this
knowledge, new wound healing strategies are emerging
to deliver growth factors to the wound bed. Surgeons
and other wound‑care specialists can use this knowledge
to identify defects and select interventions that may
promote improved wound granulation and healing.
Financial support and sponsorship
Nil.
Conicts of interest
There are no conflicts of interest.
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