![The angiogenesis and vasculogenesis processes. Recruitment of the endothelial cells from preexisting vessels plays a critical role in the regulation of angiogenesis. (1) Mobilized bone marrow endothelial progenitor cells (EPCs) with high proliferative capacity migrate and (2) home to the site of angiogenesis where (3) they deliver signals that activate pericytes, which then in turn play an active role by secreting mediators that stimulate migration and replication of local endothelial cells [46, 47]. A small amount differentiates and incorporates in the new vessel wall. EPC, endothelial progenitor cells; VEGF, vascular endothelial growth factor; erythropoietin (EPO), MMPs, nitric oxide (NO).](https://www.researchgate.net/profile/Catherine-Boisson-Vidal/publication/327542988/figure/fig2/AS:11431281169714551@1687436538884/The-angiogenesis-and-vasculogenesis-processes-Recruitment-of-the-endothelial-cells-from_Q320.jpg)
Content uploaded by Catherine Boisson-vidal
Author content
All content in this area was uploaded by Catherine Boisson-vidal on Jun 22, 2023
Content may be subject to copyright.
265
Part II
Marine Molecules for Disease Treatment/Prevention and for
Biological Research
Blue Biotechnology: Production and Use of Marine Molecules,
First Edition. Edited by Stéphane La Barre and Stephen S. Bates.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.
267
9
Use of Marine Compounds to Treat Ischemic Diseases
Catherine Boisson-Vidal
Université Paris Descartes, Unité Inserm UMR_S1140 IThEM, Facultéde Pharmacie de Paris, Sorbonne Paris Cité,
4 avenue de l’observatoire,Paris 75006, France
Abstract
e marine reservoir, with its massive biodiversity, likely harbors numerous
human drug candidates. Polysaccharides of various marine origins have shown
to be good alternatives to mammalian polysaccharides. One well-known
example is heparin, a sulfated polysaccharide used in the prevention of
thrombosis and pulmonary embolism. e oldest use of marine polysac-
charides concerns those produced by algae. ese products form the basis
for an economically important and expanding global industry. is chapter
provides a historical background to the discovery of the therapeutic potential
of these marine compounds, together with their medical and biotechnological
applications. Peripheral arterial disease (PAD) is a progressive disorder due
to atherosclerosis (narrowing of the peripheral arteries, especially in the legs).
Arterial flow is reduced or discontinuous, causing oxygen deprivation in the
underlying tissues and possible tissue necrosis. e primary aim of medical
therapy is to increase arterial flow in the affected limb in order to relieve pain,
heal trophic lesions, and avoid amputation. Anticoagulant, antithrombotic,
and antiplatelet agents are used to reduce the risk of thrombus formation.
Novel treatments such as therapeutic angiogenesis (promotion of new blood
vessel growth) are in the development phase, with promising preclinical data.
Fucoidan is a polysulfated L-fucose endowed with biological activities closely
related to its chemical composition (especially the distribution of sulfate groups
along its polyfucose backbone) and to its molecular weight. Fucoidans are
highly soluble in water, nontoxic, and non-immunogenic. Details are provided
below on its production and characterization and on the main chemical charac-
teristics that influence their biological activities. Fucoidan exhibits venous and
arterial antithrombotic properties in animal models. In animal experiments,
fucoidan promoted the formation of new blood vessels, thereby preventing
necrosis of ischemic tissue. It also recruits stem cells from bone marrow,
Blue Biotechnology: Production and Use of Marine Molecules,
First Edition. Edited by Stéphane La Barre and Stephen S. Bates.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.
268 9 Use of Marine Compounds to Treat Ischemic Diseases
further accelerating tissue healing. e cellular and molecular mechanisms
underlying fucoidan’s effects on angiogenesis are then addressed, beginning
with a brief overview of blood vessel formation. Recent advances have been
made in understanding how the interactions between these polyfucoses and
adult stem cells contribute to new blood vessel formation after ischemic injury,
notably via carbohydrates located mainly in the basement membrane and cell
surface.
9.1 History of Natural Marine Products
Drug discovery based on naturally occurring molecules in general, and on
natural marine products in particular, has been undergoing a renaissance in
recent years [1]. e relative failure of combinatorial chemistry (synthesis)-based
drug discovery has stimulated the interest of large pharmaceutical companies
in natural products that, historically, have been the source of some 50% of all
drugs. About 70% of Earth’s surface is covered by oceans, which are estimated to
contain more than 2 million species, of which only 190 000 have been identified
so far [2]. Algal extracts have long been used for medicinal purposes, notably
in Chinese traditional herbal medicine for at least 2000 years [3]. Some 400
species of seaweed are used as a source of food (especially in Asia and the Pacific
region), feedstock, medicines, fertilizers, and industrial raw materials. Some
serve as vermifuges to expel ascarids, while others are used to cure sprains,
rheumatism, goiter, cough, asthma, and dropsy. Algae have also long been used
in the prevention and treatment of cancer (Table 9.1).
Polysaccharides are the main constituents of brown and red marine algae [4]
(Figure 9.1). Seaweed cell walls are made up of various polysaccharides (cellulose,
alginates, carrageenans, and fucans; see Box 9.1) and proteins. Carbohydrates,
particularly those found on cell membranes, play critical biological roles. Defects
in either the structure or the expression of carbohydrate binding proteins are now
known to play a role in an ever-increasing number of diseases, such as vascular
disorders, viral and bacterial infections, tumor progression, and metastasis.
Marine polysaccharides also display a wide variety of biological functions, such
as extracellular matrix (ECM) organization, cell growth, and tissue maturation.
Despite their potential for drug design, very few marine-derived carbohydrates
are being developed commercially as therapeutics (see Box 9.2, Table 9.2).
More attention has been paid to the structure and composition of these marine
carbohydrates during the past 30 years, mainly because they form viscous gels
in water that remain stable in the presence of many different additives [5, 6].
Agar derived from the agarose found in the cell walls of red algae is used as a
natural vegetable gelatine. It is also extensively used as a suspending agent for
radiological solutions such as barium sulfate and as a bulk laxative that forms a
smooth and non-irritating hydrated bulk in the digestive tract. It is also used as
9.1 History of Natural Marine Products 269
Tab le 9. 1 Examples of marine-derived carbohydrate-based therapeutic molecules (GlycoMar
www.glycomar.com).
Compound Source Activity Status
Chondroitin
sulfates
Mussels
Nutraceutical
Joint repair/
maintenance
Clinical
Galactofucan
sulfated (GFS)
Seaweed
Marinova Australia
Herpes virus infections
Mobilization of stem cells
Preclinical
Fucoidan Immunity, osteoarthritis,
HSV-1, antioxidant
Phase I and II
Oligo G Alginate
AlgiPharma Norway
Cystic fibrosis Phase I and II
Heparinoid 916 Chitosan
Ocean University of
China
Atherosclerosis Phase III
Propylene glycol
alginate sodium
sulfated (PSS)
Alginate
Ocean University of
China
Antiangiocardiopathy Available in China
Propylene glycol
mannate sulfated
(PGMS)
Posterior capsular
opacification
Available in China
Heparinoid 971 Alzheimer’s disease Phase IIb
Polymannuronic
acid propyl sulfate
(PMS)
Cerebral ischemia Phase IIa
Heparinoid 911 HIV/AIDS, HBV Phase IIa
HIV/AIDS, human immunodeficiency virus infection and acquired immune deficiency syndrome;
HSV-1, herpes simplex virus type 1.
an ingredient in tablets and capsules, in order to carry and release drugs, as well
as in wound dressings and biomaterials. Alginate derivatives and carrageenans
are widely used in toothpaste, soap, ice cream, yogurt, tinned meat, fabric
printing, and a host of other applications.
Box 9.1 The Algae Compounds’ Family
Seaweeds belong to a rather ill-defined group of plants called algae. The term
“seaweed” itself has no taxonomic value but is rather a popular term used to
describe common large attached (benthic) marine algae that live on, in, or near the
seabed. They belong to the groups Chlorophyceae (4500 species), Rhodophyceae
(6500 species), and Phaeophyceae (1800 species), commonly known as green,
red, and brown algae, respectively (Figure 9.1). Rhodophyceae and Phaeophyceae
are exclusively found in oceans and seas, whereas Chlorophyceae are common in
freshwater rivers and lakes [7].
(Continued)
270 9 Use of Marine Compounds to Treat Ischemic Diseases
Box 9.1 (Continued)
Fucan is the collective name for a family of sulfated polysaccharides rich in
L-fucose and found in brown seaweed (Phaeophyceae) cell walls. These polysac-
charides were first isolated by Kylin [8] at the beginning of the twentieth century
and were originally named fucoidins. They are found in the intercellular mucilage
of marine algae, where they play a key role in wall architecture, by cross-linking
cellulose and alginates [9]. Fucans are arbitrarily divided into three main fam-
ilies: homofucans (or fucoidans), xylogucoglucuronans (or ascophyllans), and
glucuronofucoglycans (or sargassans). Fucoidans are based on L-fucose, with a
repeating structure of alternating α(1 →3) and α(1 →4) glycoside bonds and
sulfate groups at position 2 or 4, and additional monosaccharides (D-xylose,
D-galactose, and D-mannose) [11, 12]. Ascophyllans consist of a polyuronide
backbone (mainly poly-β-(1,4)-D-mannuronic acid) branched with short chains
of neutral or sulfated residues of galactose, xylose, and fucose. Sargassans
consist largely of linear chains of (1,4)-linked D-galactose or glucose branched
at C5 with L-fucosyl-3-sulfate or, occasionally, a uronic acid (usually D-glucuronic
acid).
L-
Fucose (%)
Uronic
acid (%)
Other
sugars (%)
Sulfate
groups (%) Proteins (%)
Fucoidans 50–90 <8 5–47 35–45 <4
Ascophyllans ≈25 ≈25 ≈25 ≈13 ≈12
Sargassans 25–45 ≈12 ≈36 15–21 4
Source:Liet al. 2008 [10]. Used under Creative Commons license BY 3.0.
For a given species of algae, the relative proportions of fucoidan, ascophyllan,
and sargassan depend on the tissue and many other factors such as age, habi-
tat, and season. The exact composition varies with the species. These variations
affect the properties and quality of extracts. The biological properties and quality
of fucoidan derived from rockweeds (Fucus,Ascophyllum) depend on three factors:
(i) the L-fucose composition, (ii) the amount of ascophyllan or alginate, and (iii) the
amount of phenolic compounds [13].
A high-quality fucoidan fraction has been commercially available in France
for almost 20 years, from the company Algues et Mer (http://www.algues-
et-mer.com/), being obtained by oxidative-reductive depolymerization with
H2O2of a crude high molecular weight (HMW) fucoidan. Algues et Mer is the
world’s largest producer of pure low molecular weight fucoidan (LMWF) from
Ascophyllum nodosum. It sells the product mainly for cosmetic and research
purposes.
9.1 History of Natural Marine Products 271
Figure 9.1 Colorful seaweed in rock
pool on the coast of Brittany in France.
From right bottom to the upper left:
brown algae Laminaria digitata, green
algae Ulva lactuca,redalgaePalmaria
palmata, green algae Enteromorpha
intestinalis, brown algae L. digitata,and
Fucus vesiculosus. (Courtesy of
S. La Barre.)
Box 9.2 Oligosaccharides in Use as Drugs or in Development
Carbohydrates represent the most abundant group of naturally occurring
biological molecules, being produced in billions of tons every year by plants
and photosynthetic bacteria. They are mainly produced as polysaccharides or
oligosaccharides – macromolecules composed of a large or moderate number
of monosaccharide residues, respectively. The use of carbohydrate-based drugs
is in its infancy, although there are several well-known examples: one is the
“monosaccharide-inspired” drug oseltamivir (Tamiflu®, Roche), an anti-influenza
treatment extracted from Chinese star anise. There are also two “blockbuster”
drugs: an acarbose of microbial origin sold by Bayer AG (Precose®in Europe
and China; Glucobay®in the United States) and used in the management of
type 2 diabetes mellitus and heparin, a drug derived from porcine intestinal or
bovine pulmonary mucosa and used to treat and prevent deep vein thrombosis
and pulmonary embolism, along with its low molecular weight (LMW) derivatives
such as enoxaparin (Lovenox), dalteparin (Fragmin, Pfizer Inc.), and tinzaparin
(Innohep, LEO Pharmaceutical Company). The drugs listed in Table 9.2 have
overcome some of the perceived limitations of sugar-based molecules in terms
of their synthesis, delivery, and immunogenicity. Although many of these drugs
still require intravenous or subcutaneous delivery, several are available for oral
administration. Ongoing research is seeking to improve oral bioavailability by
reducing the compounds to their smallest active components or by combining
them with other molecules such as sulodexide. Small active components can be
selected or modified to improve efficacy. No major immunogenicity problems
have been reported (except for thrombocytopenia induced by heparin), either
in animal trials or in routine clinical use of synthetic or animal-derived materials,
including those obtained from marine invertebrates.
(Continued)
272 9 Use of Marine Compounds to Treat Ischemic Diseases
Box 9.2 (Continued)
Tab le 9. 2 Examples of carbohydrate-based drugs in use or in development.
Company Compound Activity/disorder Development
Numerous Heparin and
derivatives: in
particular low
molecular weight forms
Anticoagulants Since
1940s
Astellas Pharma
(France)
Auranofin (Ridaura):
organogold
monosaccharide
Rheumatoid and
juvenile arthritis
1983
Aspri Pharma
(Canada)
Arixtra (fondaparinux):
synthetic heparin
pentasaccharide
DVT and pulmonary
embolism
1982
GlaxoSmithKline
(UK)
Zanamivir (Relenza):
modified
polysaccharide
Anti-influenza
(neuraminidase
inhibitor)
1992
Johnson & Johnson
(USA)
Topiramate (Topamax):
modified
polysaccharide
Antiepileptic 1987
Bayer (USA) Acarbose (Glucobay):
pseudo-
oligosaccharide
Type 2 diabetes
Alpha-glucosidase,
alpha-amylase
inhibitor
1990
Ortho-McNeil (USA),
Janssen
Pharmaceuticals
(Belgium), Johnson
& Johnson (USA)
Elmiron (pentosan
polysulfate)
Cystitis (interest in
development for
CJD)
1996
Alfa Wassermann
(Italy)
Sulodexide (VesselTM)Arterial
antithrombotic
Marketed
since
1980s
Merrion
Pharmaceuticals
(Ireland)
MER-102: oral uLMW
heparin
DVT Phase IIa
Sanofi-Aventis
(France)
Idrabiotaparinux:
biotinylated synthetic
heparin
pentasaccharide
DVT
Pulmonary
embolism
Phase III
Cantex
Pharmaceuticals
(USA)
PGX100: O-desulfated
heparin
Cardiac ischemia/
reperfusion injury,
COPD, used in AML
Phase
I/phase II
Progen
(Australia)
PG545: fully synthetic
heparan sulfate
mimetic
Antiangiogenic
Antimetastatic
Phase I
(Continued)
9.1 History of Natural Marine Products 273
Tab le 9. 2 (Continued)
Company Compound Activity/disorder Development
PI-88: phospho-
mannopentose
sulfate
Antiangiogenic
Antimetastatic
Phase III
GlycoMimetics Inc.
(USA)
GMI1070 Vaso-occlusive crisis
of sickle cell disease
Phase I
GM1010/1011/1043 Anti-inflammatory/
pulmonary
inflammation
Preclinical
GlycoMar/Verona
Pharma (UK)
GLY145:
glycosaminoglycan
Anti-inflammatory Preclinical
AML, acute myeloid leukemia; CJD, Creutzfeldt–Jakob disease; COPD, chronic
obstructive pulmonary; DVT, deep venous thrombosis.
Source: GlycoMar www.glycomar.com.
Box 9.3 Fucoidan, A Promising Product?
Letourneur’s team showed that fucoidan could serve as a platform for the
development of thrombus-targeting contrast agents. A LMW fucoidan fraction
prevents P-selectin binding to sialyl Lewis X with an IC(50) of 20 nM, as compared
with 400 nM for heparin and >25000 nM for dextran sulfate. It exhibits the
highest affinity for immobilized P-selectin compared with these polysaccharides.
As a consequence, fucoidan binds to platelets with an intensity that depends
on the level of platelet activation [14]. The interaction is highly specific, as
shown by competition assays between fucoidan and an anti-P-selectin antibody.
Letourneur et al. radiolabeled fucoidan with 99mTc for scintigraphy and with
ultrasmall superparamagnetic iron oxide particles (USPIO agents) for magnetic
resonance imaging (MRI). This allowed the detection of areas of platelet activation
and subsequent thrombus formation in vivo [15]. USPIO–LMWF conjugates were
shown to target P-selectin. Flow cytometry showed interaction with activated
platelets in human whole blood in vivo [16]. USPIO-Fuco is now being developed
as an MRI contrast agent for thrombus imaging (http://www.google.com/patents/
WO2010116209A1?cl=en; http://www.google.ch/patents/US20140134102).
Research on fucoidans extracted from brown algae has steadily increased
in recent years. Despite a wide range of biological activities and the lack of
oral toxicity [17, 18], fucoidans remain relatively unexploited as a source of
medicines because of its heterogeneity. Indeed, they cannot yet be synthesized,
and obtaining reproducible batches remains highly challenging. Although they
have been commercially available for half a century, differences in their chemical
composition and molecular weight between batches have limited pharmaceutical
development. e international fucoidan market is nonetheless growing.
274 9 Use of Marine Compounds to Treat Ischemic Diseases
9.2 Peripheral Arterial Disease and Cardiovascular
Risks: Treatments and Unmet Needs
Peripheral arterial disease (PAD) is characterized by chronic obstruction of arter-
ies that supply tissues. e affected tissues suffer from ischemia and can develop
necrosis if blood circulation is not quickly reestablished, with a risk of losing
a limb or its function. PAD is a frequent disease affecting between 1% and 3%
of people older than 60 years [19–22]. It results from atherosclerosis, throm-
bus formation, or inflammatory processes and develops in several stages. Crit-
ical limb ischemia (CLI) is the end stage of PAD. Severe obstruction of blood
flow results in ischemic pain at rest, ulcers, and a significant risk of limb loss.
Epidemiological studies show that the incidence of PAD increases with age, up
to 5% beyond 70 years. PAD progresses slowly, but 20–30% of patients even-
tually develop CLI (500–1000 per million inhabitants) requiring revasculariza-
tion or amputation (more than 5000 cases yearly in France [23]). About 18%
of patients die during the year after CLI onset, and 26% require amputation.
Surviving patients need special care that is both heavy and costly.
PAD affects the blood vessels supplying the arms and legs. Normal arteries are
lined by a thin layer of endothelial cells (endothelium), which help to keep blood
flowing. High blood pressure, smoking, and high cholesterol can damage the
endothelium and lead to the formation of atherosclerotic plaque or atheroma.
e latter results from the invasion and accumulation of white blood cells and
proliferation of intimal smooth muscle cells that create a “fibrofatty” plaque.
is plaque can stay fixed to the artery wall and slowly grow, or may suddenly
rupture, triggering platelet activation and aggregation on their exposed surface
(Figure 9.2). Plaque rupture can add coronary artery or cerebrovascular disease
to the underlying PAD.
Immobilization, chronic venous insufficiency, and venous blood stasis in the leg
increase the risk of vein thrombosis associated with PAD [24]. rombus forma-
tion disrupts blood flow, leading to concerted interaction of activated endothe-
lium with neutrophils and platelets, mediated mainly by P-selectin expression on
endothelial cells.
Treatment is aimed at correcting the underlying causes of ischemia and at
improving the vascularization of affected tissues in order to prevent gangrene
and limb loss and to reduce the risk of heart attack and stroke [19, 25].
9.2.1 Prevention of Disease Progression
Treatment focuses first on modifiable risk factors such as hypertension, dyslipi-
demia, and diabetes mellitus through lifestyle modifications [19, 20, 25]. Smoking
cessation and exercise programs are proposed to alleviate symptoms. Drug treat-
ment may be prescribed: (i) anticoagulants and/or antiplatelet/antithrombotic
drugs (aspirin, clopidogrel) to prevent thrombus formation due to slow blood
flow in microvessels [26], (ii) vasodilatory drugs to prevent platelet aggregation
(patients with PAD have a systemic endothelial dysfunction associated with
impaired vasodilation and enhanced platelet aggregation [27], (iii) antihyperten-
sive drugs, and (iv) cholesterol-lowering drugs [28]. ere is a strong correlation
between high low-density lipoprotein cholesterol levels and PAD symptoms.
9.2 Peripheral Arterial Disease and Cardiovascular Risks: Treatments and Unmet Needs 275
Normal artery wall
Gradual redirection
of flow
Insufficient
angiogenesis
Insufficient
colateral growth
Hypoxia
Endothelial
dysfunction
No tissue
regeneration
Plaque rupture Thrombosis
Figure 9.2 Peripheral arterial disease in the lower limb is characterized by chronic obstruction
of the arteries supplying the leg, gradually leading to CLI. High blood pressure, smoking, and
high cholesterol can damage the endothelium and lead to the development of a fatty streak
under the endothelium. An unstable plaque develops with a fatty core that can sometimes
rupture into the bloodstream. Clotting of the blood begins at the site of the plaque rupture.
The artery becomes partially blocked and blood flow is diminished. Accordingly, there is
insufficient blood flow to redirect toward the collaterals. Furthermore, acute arterial occlusion
leads to distal tissue hypoxia and causes the activation of an inflammatory response against
ischemic tissue injury owing to endothelium dysfunction and several factors. The insufficient
collaterals and angiogenic signaling limit tissue regeneration and can lead to necrosis and loss
of tissue function if revascularization is not reestablished.
9.2.2 Anticoagulation
Despite its disadvantages, heparin has been the polysaccharide most widely
used to treat venous thrombosis for the past 70 years [29]. Heparin is a highly sul-
fated glycosaminoglycan (GAG): 80% of glucosamine residues are N-sulfated, and
O-sulfate groups are more abundant than N-sulfate groups. is mucopolysac-
charide mainly exerts its anticoagulant activity by enhancing the inhibitory
potency of the plasma proteins antithrombin (AT) and heparin cofactor II
(HCII), which inhibit serine proteases involved in the coagulation pathway, such
as thrombin and factor Xa (FXa) [30]. e anticoagulant activity of heparin is due
to a specific pentasaccharide sequence that binds with high affinity to AT [31].
9.2.3 Surgical Revascularization
Between 40% and 60% of patients with CLI may need surgery to inject throm-
bolytic drugs (urokinase or rt-PA to dissolve a thrombus) or to bypass a blocked
or narrowed artery with an artificial vessel to improve the microcirculation
[25, 32]. Balloon angioplasty and stenting are proposed to patients with short
arterial occlusions, as a first-line attempt at limb salvage [32]. When surgical
276 9 Use of Marine Compounds to Treat Ischemic Diseases
revascularization is not feasible, amputation is often the only therapeutic option.
No available treatment has so far been shown to reduce the amputation rate at
6 months in patients with CLI [33].
9.2.4 Novel Therapies: Therapeutic Angiogenesis
Much research is focused on ways of promoting the development of new
blood vessels (therapeutic angiogenesis), including gene therapy [34–37] and
cell therapy [34, 38, 39]. Despite encouraging phase II trials, however, large
prospective double-blind trials of a genetic drug coding for a proangiogenic
growth factor have shown little improvement in the risk of amputation or death
[19, 25, 37, 40, 41].
9.2.4.1 Cell Therapy
Cell therapy, which is more promising, consists of local intramuscular injections
of autologous bone marrow-derived mononuclear cells into the ischemic limb.
Several randomized trials are underway to test the safety and efficacy of stem
cells in the treatment of PAD [21, 39, 42–44].
9.2.4.2 How Do New Blood Vessels Form?
e formation of new blood vessels involves at least three distinct biological
processes [45] (Figure 9.3). Angiogenesis refers to the formation of new vessels
by “sprouting” from a preexisting vascular network. Arteriogenesis refers to an
increase in the wall thickness and luminal diameter of newly formed vessels
via the recruitment of perivascular and smooth muscle cells. Vasculogenesis is
theemergenceofbloodvesselsde novo from vascular endothelial progenitor
cells (EPCs) known as angioblasts, which differentiate into endothelial cells
[48]. ese three processes probably occur simultaneously during new vessel
formation in the lower limbs of patients with PAD. Occlusion of an artery causes
tissue hypoxia, which is a strong stimulus for angiogenesis [49]. Collateral vessels
develop physiologically in these patients, leading to an increased microvessel
density in affected muscles, which can partially compensate for occlusion of the
artery but often fails to restore normal flow [45, 50].
e discovery in 1997 that adult peripheral blood contains circulating EPCs
capable of differentiating into endothelial cells provided new insights into
vascular biology and opened up exciting therapeutic vistas [46, 51–53]. Asa-
hara’s team showed the contribution of these cells to blood vessel formation
at sites of active angiogenesis and obtained tissue regeneration by infusion of
autologous EPCs after ischemic injury [54], leading to the concept of therapeutic
angiogenesis [55]. ese bone marrow-derived angiogenic cells are mobilized
to the general circulation and then recruited to sites of ischemia in response
to various stimuli produced by the local inflammatory response, including
growth factors and cytokines such as vascular endothelial growth factor (VEGF),
angiopoietin-1, granulocyte macrophage colony-stimulating factor (GM-CSF),
and stromal-derived factor-1 (SDF-1) (Figure 9.3). ese angiogenic cells also
participate in the development of new blood vessels by releasing factors that
attract and activate pericytes and smooth muscle cells, which in turn secrete
9.2 Peripheral Arterial Disease and Cardiovascular Risks: Treatments and Unmet Needs 277
Angiogenesis Vasculogenesis
Sprouting from preexisting
vessels
Mature
endothelial cells
Bone marrow
derived progenitor
cells
3 Effect of EPC:
- Incorporation in new blood vessels
1 mobilization :
VEGF, EPO,
MMPs
2 Homing : VEGF,
integrins,
P-selectin,
E-selectin
- Release of paracrine factors
Growth factors
Cytokines
NO
Hypoxia
Incorporation of EPCs
Figure 9.3 The angiogenesis and vasculogenesis processes. Recruitment of the endothelial
cells from preexisting vessels plays a critical role in the regulation of angiogenesis.
(1) Mobilized bone marrow endothelial progenitor cells (EPCs) with high proliferative capacity
migrate and (2) home to the site of angiogenesis where (3) they deliver signals that activate
pericytes, which then in turn play an active role by secreting mediators that stimulate
migration and replication of local endothelial cells [46, 47]. A small amount differentiates and
incorporates in the new vessel wall. EPC, endothelial progenitor cells; VEGF, vascular
endothelial growth factor; erythropoietin (EPO), MMPs, nitric oxide (NO).
soluble mediators that stimulate the migration and differentiation of local
endothelial cells [56].
e ability of EPCs to mobilize and migrate to ischemic sites thus holds promise
for this therapeutic approach [46, 57] (Figure 9.4). In animals, these immature
cells can be mobilized from bone marrow into the circulation by treatment with
a variety of compounds, including marine polysaccharides [13, 58–60], but this
process becomes less efficient with age [61]. Reduced EPC numbers and functions
are indicators of severe endothelial lesions in patients with PAD [46, 62, 63].
Stem cells can be derived from bone marrow, peripheral blood, or cord
blood, but clinical applications have been hindered by ethical concerns and by
difficulties in harvesting these cells or in safely and efficiently expanding them.
Infusion of these bone marrow stem cells improved new blood vessel formation
in animal models of PAD, enhancing collateral vessel formation and blood per-
fusion and permitting limb salvage even after complete vessel occlusion [46, 51,
53, 64]. However, proangiogenic approaches have so far given disappointing
results in patients with CLI, owing partly to poor engraftment and limited
278 9 Use of Marine Compounds to Treat Ischemic Diseases
Injection
statins,
GM-CSF, etc.
Isolation from peripheral blood
by density gradient
centrifugation and expansion Priming with
SDF1, VEGF,
Fucoidans,
Hypoxia, etc.
Animal model of PAD
Figure 9.4 Activation of local angiogenesis is a promising approach to patients with critical
limb ischemia with a high risk of amputation. Stem/progenitors cells isolated from bone
marrow or peripheral blood can be pre-activated with SDF-1, VEGF, fucoidan, hypoxia, and so
on or modified to improve survival, homing, functional engraftment, and functional activity
(e.g., differentiation) before being infused or injected in patients. Infusion of these in vitro
expanded bone marrow cells enhances neovascularization in animal models of hindlimb
ischemia.
differentiation/survival at the ischemic site [19, 21, 25, 61]. Indeed, only a very
small fraction of transplanted cells are incorporated into new vessels. EPCs iso-
lated from peripheral blood of CLI patients have reduced proliferative capacity
and increased apoptotic activity and secrete only small amounts of VEGF.
9.3 Chemistry
9.3.1 Extraction and Preparation of Low Molecular Weight
Fucoidan Fractions
Marine algae are immediately dried to reduce spoilage and chemical degradation
and are then stored at room temperature. Various extraction methods have been
described [11, 12, 65–68]. Isolation and purification of fucans generally involves
aqueous extraction (hot water, dilute acid (mainly hydrochloric or sulfuric acid
(pH 2.0) or alkalis), sometimes with extracting agents; fractional precipitation
with ethanol, lead salts, calcium salts, or quaternary ammonium salts; or frac-
tionation on ion exchange columns [66, 69]. Partial fucan degradation may occur,
especially at high temperatures and acidic pH. Maceration with dilute hydrochlo-
ric acid or sulfuric acid removes any soluble mineral salts. Phenolic compounds
can be removed by preheating with formaldehyde or by extraction at pH 6–7.
9.4 Biological Properties 279
e extracts thus obtained contain other polysaccharides (laminaran and
alginic acid) and LMW substances (colored materials, phenolic compounds,
mannitol, D-glucose, and, in some cases, myo-inositol), as well as fucans of
(ascophyllan and sargassan) [9, 12]. Pretreatment with 80% aqueous ethanol
before extraction can remove these LMW compounds. Crude fractions are
purified and fractionated with ethanol, ion exchange chromatography, or gel
filtration chromatography. e purification yield varies with the pH of the
extracting medium, the temperature, and the extraction time.
LMWF can then be obtained by acidic hydrolysis [67] or oxidative-reductive
depolymerization [70]. e oxidative-reductive process reduces the molecular
weight (depolymerization) under mild conditions (pH 7, ambient temperature),
with high yields.
9.3.2 Structural Determination
Fucoidan being a natural product, it is necessary to ensure the characterization
and reproducibility of the bioactive fraction, although small variations in chem-
ical composition or molecular weight do not significantly affect its biological
properties. e homogeneity of fucoidan preparations can be determined by gel
filtration, ion exchange chromatography, and electrophoresis. Fucosidases and
enzymes that degrade polyanionic polysaccharides are used to establish fucoidan
structure and structure–activity relationships [71].
e type of fucoidan, its sulfation and molecular weight, and the conformation
of its sugar residues all vary across seaweed species and with the period and place
of their harvest [12, 65, 72]. Within a given species, the chemical composition of
the fucoidan extract depends on the extraction conditions. Free fucans released
upon cell wall isolation are similar in composition to fucoidan in Pelvetia canalic-
ulata,Ascophyllum nodosum,Fucus vesiculosus,andLaminaria digitata and to
sargassan in Sargassum muticum [73].
Fucoidan is mainly composed of α(1 →3) and α(1 →4) fucosyl units, usu-
ally sulfated at position 2 or 4 (rarely C3), with additional monosaccharides
(D-xylose, D-galactose, and D-mannose) [10–12, 74]. Chevolot et al. [75] reported
that the predominant repeating structure of fucoidan from A. nodosum is
[(1 →3)-α-L-Fuc(2SO3−)-(1 →4)-α-L-Fuc(2,3diSO3−)-(1)]n[75]. e respective
molar ratio of sulfate groups to total sugars (including uronic acid) is 3 : 2,
suggesting that three moles of sulfate may be attached mainly to two fucose
residues in the polysaccharides.
9.4 Biological Properties
9.4.1 Marine Polysaccharides Exhibit Anticoagulant Activity
Extracts from over 60 species of brown, red, and green seaweed have been
reported to exhibit anticoagulant capacity [5, 6, 65]. e main active compo-
nents are sulfated polysaccharides [76], although not all sulfated carbohydrates
possess anticoagulant activity [12, 77, 78]. Extracts of brown marine algae have
higher anticoagulant activity than extracts of red and green marine algae.
280 9 Use of Marine Compounds to Treat Ischemic Diseases
9.4.1.1 Principle of Anticoagulation
Anticoagulation is achieved mainly by inhibiting thrombin and FXa, two serine
proteases. is can be done by enhancing the activity of the main physiolog-
ical serine protease inhibitors, namely, AT and HCII. In vitro, heparin greatly
accelerates the rate of thrombin inhibition both by AT and by HCII [29, 30]. e
mechanism of heparin-catalyzed thrombin inhibition by AT and HCII involves
the formation of a ternary complex between heparin, proteinase inhibitor, and
proteinase [31].
9.4.1.2 Marine Polysaccharides Have Potent Anticoagulant Properties
Carrageenans from red algae have been studied more extensively than fucans
[65, 79]. e mechanism for their anticoagulant activity does not involve AT.
Even at high dilutions, they exhibit significant anticoagulant activity. However,
their clinical use is limited by their immunogenicity and tendency to gel due to
their structural heterogeneity and very HMW. Agar has similar properties [80].
9.4.1.3 Fucoidan Exhibits Venous and Arterial Antithrombotic Properties
with No Hemorrhagic Risk
In 1957, Springer et al. [66] reported that fucans extracted from the brown marine
alga F. vesiculosus had anticoagulant activity in vitro and in vivo [66]. is activ-
ity was neutralized by protamine, showing the contribution of sulfate groups.
Fucoidan was added to the group of heparinoids. e anticoagulant activity of
fucoidan correlates with the chemical composition of the different fractions [10,
17, 65, 81]. Anticoagulant activity is mainly related to a heparin-like effect on
AT and HCII and to direct interaction with thrombin [82, 83] (Figure 9.5). It
correlates mainly with the molecular weight distribution, the sulfate group con-
tent, and, probably, the structure of the fucoidan. e negative charge density of
fucoidan is required for the expression of its AT activity mediated by HCII.
Fucoidan exhibits venous and arterial antithrombotic properties in exper-
imental animals [10, 72, 84] and, contrary to heparin, does not significantly
increase the risk of bleeding. It increases the AT activity of the protease nexin-1
(PN-1), a pericellular serpin expressed by vascular cells, and thereby significantly
reduces circulating levels of thrombin by catalyzing its inhibition [85]. Fucoidan
also promotes fibrinolysis by potentiating plasminogen activators and reducing
plasminogen activator inhibitor-1 (PAI-1) release by endothelial cells [86, 87].
9.4.2 Marine Polysaccharides Have Angiogenic Properties
Several sulfated polysaccharides, including heparin, structural mimics of hep-
aran sulfate, and carrageenans, have been found to inhibit angiogenesis in vitro,
usually through a direct effect on endothelial cells when added to the experi-
mental medium [13, 29, 81]. ese properties are due in part to the capacity of
these polyanions to bind to the heparin binding site of proangiogenic growth fac-
tors (mainly VEGF and basic fibroblast growth factor 2 (FGF2)) via their sulfate
groups. Commercial HMW fucoidan reduced neov ascularization and suppressed
tumor angiogenesis in experimental models by triggering endothelial cell apop-
tosis [74]. ese effects are related to both the sulfation ratio and the molecular
weight.
9.4 Biological Properties 281
TF
FV
Fibrinogen
Prothrombin
tPA
Fuc PAI Thrombus
Plasminogen
Plasmin
Fibrin clot
AT
AT
HCII
IIa
FX
Fuc Fuc
FXa
FXa
FVIIa
Fuc
Fuc
Figure 9.5 Fucoidan (Fuc) can prevent thrombosis by catalyzing thrombin inhibition and
promoting fibrinolysis. It reduces circulating level of thrombin by catalyzing its inhibition:
tissue factor (TF) bound to the endothelium initiates the coagulation cascade. It binds to
activate circulating factor VII (FVIIa) and catalyzes the activation of factor X. Then factor Xa
(FXa) binds to factor V (FV) present on the phospholipid surface of platelets or endothelial cells
to catalyze the conversion of prothrombin to activate thrombin (IIa). Thrombin cleaves
fibrinogen to form fibrin that leads to the formation of fibrin clot and then a thrombus. The
propagation of the cross-linked fibrin clot is limited by plasmin, which cleaves fibrin.
Endothelial cells secrete tissue plasminogen activator (tPA) to activate plasmin and control the
expansion of the clot on the injured endothelial cell surface. Fuc is a direct inhibitor of
thrombin and catalyzes the inhibition of serine proteases Xa and thrombin by antithrombin
(AT) and the inhibition of thrombin by heparin cofactor II (HCII). It promotes fibrinolysis by
potentiating plasminogen activators such as tPA and reducing plasminogen activator
inhibitor-1 (PAI-1) release by endothelial cells.
9.4.2.1 Contrary to Other Polysaccharides, Fucoidan Potentiates Angiogenesis
In Vitro and In Vivo
Fucoidan pretreatment of cultured human umbilical vein endothelial cells
(HUVEC) or EPCs for 36–72 h enhances FGF-2-dependent angiogenesis
in vitro [88, 89], while the addition of fucoidan directly to the culture medium
inhibits growth factor-induced vascular tube formation by endothelial cells
[13, 90]. Fucoidan prestimulation enhances various angiogenic processes,
namely, cell recruitment to ischemic tissue via enhanced EPC adhesion to acti-
vated endothelium; MMP-9 matrix metalloproteinase secretion; extravasation;
and differentiation into a vascular network (Figure 9.6). e beneficial effect of
282 9 Use of Marine Compounds to Treat Ischemic Diseases
5 Recruitment of
stem cells
4 Adhesion on ECM
or activated endothelium
3 Tube formation
1 Proliferation
2 Directional
migration
100
% Cell mobilization
SDF-1 ng mL–1
CTRL
A
B
LMWF VEGF
CTRL LMWF VEGF
CTRL
ABC
FGF2
60
3000
Control EPCs
VEGF
0
Cell number
2.0 10
5
1.5 10
5
0.5 10
5
0
0.01 0.1
Fucoidan concentration (μg mL–1)
1 10 100 1000
400
300
200
100
Chemoattractant (ng mL–1)
lower chamber 40 40
VEGF--
Fucoidan-
pretreated
EPCs
2500
Total lenght of tubes structures
(% control)
2000
1500
1000
500
0
FGF-2 + Fucoidan
FGF-2
Control
50
40
30
20
10
--
Cell transmigration (% EPC
without chemoattractant)
0
Chemoattractant (ng mL–1)
lower chamber
VEGF
40
EPC EPC + Fuc
40
VEGF
FGF2 + Fuc
90
80
70
60
50
40
30
20
10
0
9
8
7
6
5
4
3
2
1
0
Figure 9.6 Fucoidan induces a proangiogenic phenotype in human EPC. (1) Fucoidan
enhances EPC proliferation in a concentration-dependent manner starting at 1 μgmL
−1.
(2) Fucoidan pretreatment promotes EPC motility (no chemoattractant) and enhances EPC
chemotaxis toward VEGF. 3. Fucoidan pretreatment enhances basic fibroblast growth factor 2
(FGF2)-induced vascular tube formation by EPC: (3A) EPCs do not form tubular structures in
control medium, (3B) 18 h after seeding, FGF2-pretreated cells are elongated and
interconnected, and (3C) the tubular network is significantly more extensive in the presence of
FGF-2 and fucoidan. (4) Fucoidan pretreatment enhances EPC adhesion on activated
endothelium under static and dynamic conditions and their extravasation toward VEGFs
(40 ng mL−1). (5) Fucoidan induces mobilization of immature CD34+CD31+CD45−murine
progenitors. Wild mice were intraperitoneally injected with PBS (negative control, CTRL),
5mgkg
−1of low molecular weight fucoidan (LMWF), or 0.5 μgkg
−1VEGF (positive control,
VEGF) and bled 30 min after the injection. Blood was assayed for CD34+CD31+CD45−cells, and
plasma samples were analyzed for SDF-1 concentration by enzyme-linked immunosorbent
assay test. (5A) Progenitor mobilization after intraperitoneal injection of PBS (negative CTRL),
LMWF, or VEGF. (5B) SDF-1 level in mice plasma after intraperitoneal injection of fucoidan
compared with controls. Results are expressed as the means ±SEM *p<0.05; **p<0.01;
***p<0.001.
9.4 Biological Properties 283
(a)
PBS EPC + Fuc
NI
CTRL
CTRL
NI
Isch
ECFC
Necrotic
area
Ischemic
area
Preserved
area
ECFC + Fuc
ECFC ECFC + Fuc
CTRL
NI
Isch
ECFC ECFC + Fuc
CTRL
p = 0.062
p = 0.281
p = 0.151
ECFC ECFC+Fuc
CTRL ECFC ECFC + Fuc
(c)
(f)
(g)
(d)
(e)
Isch NI
1.0
60 70
60
50
40
30
20
10
0
50
40
30
Necrotic area/ Total area ratio
(Operated limb)
Preserved area/ Total area ratio
(Operated limb)
20
10
0PBS PBS
Preserved area
Fuc
80%
CTRL
20% CTRL
92%
CTRL
88%
Fuc
8% Fuc
12%
Ischemic area Necrotic area
EPC EPCEPC+Fuc EPC + Fuc
0.9
0.8
0.7
0.6
0.5
Clinical necrosis score
Isch leg
0.4
0.3
0.2
0.1
0
1.0
*
##
∗∗
∗
∗∗
∗∗∗
∗
#
0.9
0.8
0.7
0.6
0.5
Foot perfusion
(Isch/N Isch)
0.4
0.3
0.2
0.1
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
Angiographic score
(Isch/N Isch leg ration)
0.2
0
0
Isch
(b)
Figure 9.7 Fucoidan protects ischemic tissue against necrosis. Fucoidan pretreatment of
cultured EPC (endothelial colony-forming cells, ECFC) increases neovascularization in hindlimb
ischemia of mice with PAD 14 days after artery ligation and intravenous injection of pretreated
ECFC. Macroscopic aspects of ECFC-injected mice (a) and fucoidan-ECFC-injected mice (b).
Cumulative incidence of clinical necrosis (c), foot perfusion (d), and quantitative analysis of
angiographic score (e) of normal saline (CTRL, ◾), untreated ECFC (ECFC, ◽), and
fucoidan-stimulated ECFC (ECFC, green ◽) transplanted mice. Hematoxylin and eosin staining
of the same distal gastrocnemius muscle sections on day 14 and quantification of
histologically preserved area, ischemic infiltrated area, and necrotic area type surface (f). The
surface of each area type is reported as a percentage of the entire histological section surface.
(g) To evaluate the effect of fucoidan on critical ischemia, two groups of animals underwent
the surgical procedure and received two intramuscular injections 1 and 2days after surgery of
normal saline and fucoidan solution (15 mg kg −1Fuc). Quantification of histologically
preserved area, ischemic infiltrated area, and necrotic area type surface 14 days after surgery
and intramuscular bolus administration of fucoidan. Values are expressed as means
±SEM (n=15) *p<0.05, **p<0.01, and ***p<0.001 versus normal saline-injected mice
(CTRL). NI, non-ischemic hindlimb; isch, ischemic hindlimb.
EPC infusion in a mouse model of hindlimb ischemia was significantly amplified
by fucoidan stimulation, preventing tissue necrosis [91] (Figure 9.7). Fucoidan
can also promote new blood vessel formation when infused intramuscularly,
either alone or together with the proangiogenic growth factor FGF-2 [92].
Fucoidan induced an 87% reduction in necrosis and a 75% improvement in
muscle preservation on day 14, as compared with saline alone [91]. Intravenous
fucoidan infusion had protective effects in rat models of myocardial ischemia
[93]. is protection was associated with enhanced neoangiogenesis and a
284 9 Use of Marine Compounds to Treat Ischemic Diseases
reduction in rhabdomyolysis. During ischemia, muscle destruction causes
permeabilization of muscle fibers and the release of their contents, including
enzymes such as CPK, into the bloodstream. Bolus administration of fucoidan
induced a 70% reduction in CPK activity as compared with normal saline. None
of the mice treated with fucoidan presented necrosis of the fingers or legs,
contrary to saline-treated controls [91].
Fucoidan also inhibits smooth muscle cell proliferation in vitro [94, 95] and in
vivo [96, 97]. Its injection prevented neointima formation in an injured thoracic
aorta rat model and after stenting in the rabbit iliac artery angioplasty model.
Fucoidan stimulated the formation of an endothelial cell lining in vascular
allografts after 1 month of treatment [98]. Injection of crude heparin and LMW
heparin in the same experimental conditions did not induce angiogenesis [13, 92].
9.4.2.2 Injections of LMW Fucoidan Induce Rapid Mobilization of Stem Cells
from Bone Marrow
Intraperitoneal (IP) or intramuscular injections of LMWF (5 mg kg−1day−1)
induce rapid mobilization of immature progenitors from bone marrow, about
as potently as VEGF (0.5 μgkg
−1day−1) [13]. Circulating numbers of progenitor
cells are significantly increased in the peripheral blood of fucoidan-treated
animals. Stem cell mobilization is also observed after oral fucoidan ingestion
[17, 99]. is mobilization correlates with the release of GAG-bound SDF-1
from its tissue storage sites into the circulation on fucoidan administration.
Tissue ischemia is accompanied by inflammation, and fucoidan has been
shown to possess anti-inflammatory properties [10, 12, 17, 100]. Daily subcuta-
neous injection of fucoidan protected parenchymal tissue against inflammatory
processes and myofibroblastic remodeling in a rat cardiac allograft model [101].
It reduced neutrophil, macrophage, and CD4+T-cell infiltration at sites of
inflammation and improved myocardial status after ischemia/reperfusion injury
[17]. ese anti-inflammatory activities correlated with a reduction in tissue
expression of transforming growth factor beta (TGFβ) and IL-10. Fucoidan
downregulated the expression of inducible iNOS (that inhibits nitric oxide
production) and cyclooxygenase 2 (COX2) protein and mRNA levels.
9.4.3 What is the Fucoidan Mechanism of Action?
9.4.3.1 Cellular and Molecular Mechanisms Underlying Fucoidan
Proangiogenic Activity
e effects of fucoidan appear to be mediated mainly by its charge density,
molecular weight, and degree of sulfation, rather than by a specific carbohydrate
structure [75]. Fucoidan shares several characteristics with heparan sulfate
proteoglycans (HSPGs). e structural requirements for its interaction with
coagulation factors and target proteases depend on the proportions and/or
distribution of sulfated groups along the fucose backbone and on the saccharide
chain composition [102]. Fucoidan fractions presenting slight differences in
their sulfation pattern differ in their anticoagulant and antithrombotic activities
[72, 103]. Sulfation is critical for fucoidan activity, but sulfate groups alone are
unlikely to be responsible for the observed biological properties. e fucosyl
9.4 Biological Properties 285
backbone may also be important. Fucosylated chondroitin sulfate species, made
up of alternating β-D-glucuronic acid and N-acetyl-β-D-galactosamine units with
branches of sulfated fucose, enhance FGF-2-induced tubular morphogenesis
in vitro andalsotriggeranincreaseinbloodSDF-1levelsandimmaturecell
mobilization. Like fucoidan, these chondroitin sulfate species that bear sulfated
fucose chains express antithrombotic activity in experimental animals with
venous and arterial thrombosis. Branched sulfated fucoses are the key motif
responsible for this activity, as shown by comparing native and chemically
modified defucosylated and/or desulfated chondroitins. e number of sulfate
groups, and the nature of the molecular backbone significantly influence the
biological properties of these compounds [102].
9.4.3.2 Fucoidan Modulates the Biological Activity of Angiogenic Heparin
Binding Proteins
Of particular interest, fucoidan partially restores the adhesion of cultured pro-
genitor cells to activated endothelium after their treatment with enzymes that
remove HSPGs from the cell surface [91]. Owing to its ionic structure, fucoidan
mimics some properties of GAGs and can bind and modulate the activity of a
large number of proteins, including proteases, cytokines, and growth factors. As
a result, fucoidan affects many biological activities in vitro, including coagulation,
inflammation, viral infection, fertilization, and angiogenesis [12, 17, 74, 104].
9.4.3.3 Fucoidan May Act as a Direct Growth Factor Signal Transducer
e proangiogenic activity of fucoidan is linked to its direct interaction with cell
membranes. Fucoidan binds to the EPC outer membrane at displaceable binding
sites and is then internalized by endocytosis. ere it can enhance local angio-
genesis through the transduction of intracellular signals required to induce the
proangiogenic phenotype. Endothelial cell surface-bound fucoidan could also act
as a coreceptor for angiogenic growth factors. e putative fucoidan receptor
function might involve a carbohydrate binding domain that interacts with the
fucoidan carbohydrate backbone. Fucoidan might thus interact with macrophage
TLR4 and SR receptors that recognize carbohydrate structures [105]. Its bind-
ing to these receptors induces several signal transduction events (p38, MAPK,
ERK1/2, and SAPK/JNK activation) that lead to macrophage activation. Because
of its spatial structure, it can mimic the clustering of sulfated, sialylated, and
fucosylated oligosaccharides on the cell surface and can provide the appropriate
structural backbone to bind selectins with high affinity [14, 106]. It is also able
to interact with other receptors on the cell membrane, such as CD44 (hyaluronic
acid receptor), αM(CD11b), β2(CD18), and even αMβ2(macrophage antigen-1
(Mac-1)) [91].
Fucoidan binds to the cell membrane and can thereby induce cellular responses
analogous to signal transduction by growth factors. It is thus able to modulate
the expression of genes involved at different stages of neovessel formation, such
as cell migration, cytoskeleton organization, cell mobilization, and homing [107].
Pseudotube formation induced by fucoidan correlates in vitro with overex-
pression of the α6 integrin subunit on the endothelial cell surface [89, 107]. is
α6 overexpression results from transcriptional upregulation. e integrin α6
286 9 Use of Marine Compounds to Treat Ischemic Diseases
chain, when assembled with integrin β1orβ4 subunits, forms major receptors
for laminin. Several results show that α6 integrins function in vivo as homing
receptors for hematopoietic stem cells and progenitor cells [108, 109]. e
same integrins also promote vascular repair. e effect of fucoidan on α6
expression on the cell membrane would strongly enhance stem cell migration
and mobilization in vivo. HSPGs can transduce extracellular signals in a manner
analogous to signal transduction by growth factor receptors. Fucoidan may act
as a direct signal transducer. It is conceivable that membrane-bound fucoidan
may participate directly in signal transduction in response to growth factor
binding by interacting directly with its receptors. e idea that coreceptors may
act as signaling molecules independent of their role as receptor binding partners
is beginning to gain ground.
9.4.4 How Does Fucoidan Act In Vivo?
In vivo, fucoidan can bind to numerous proteins. It actively modulates coagu-
lation and can reduce circulating levels of thrombin by catalyzing its inhibition
[10, 12, 17, 65, 76, 81, 83]. Lower thrombin generation would attenuate thrombus
formation, thereby enabling macrophages and EPCs to migrate to the lesion and
ensure reperfusion. GAGs have a crucial role in mediating stem cell recruitment
and homing to ischemic tissues. GAGs play a key role in sequestering a variety
of proteins that regulate the proliferation, differentiation, and trafficking of stem
cells between the bone marrow and the peripheral blood.
Several lines of evidence suggest that fucoidan acts through SDF-1. Intramus-
cular fucoidan injections increase plasma levels of SDF-1 and thus promote stem
cell mobilization [13, 59, 92]. SDF-1 is anchored to HSPGs present at the surface
of stromal cells and endothelial cells and on the ECM. Fucoidan can specifically
displace sequestered SDF-1 from its HSPG anchors on the endothelial surface
and can thereby contribute to its release into the circulation. According to
Sweeney, the amount of SDF-1 thus released into the circulation is sufficient to
trigger the observed cell mobilization [59]. In addition to SDF-1, levels of other
chemokines (IL-6, IL-8) and cytokines (granulocyte CSF, macrophage CSF) are
also increased after fucoidan treatment, possibly owing to their release from
HSPGs.
In his excellent review of sulfated fucans and galactans, Pomin [110] forwards
a mechanism to explain the anti-inflammatory properties of marine polysaccha-
rides that could also account for the impact of fucoidan on EPC recruitment.
e molecular mechanisms behind these proangiogenic properties would involve
(Figure 9.8) (i) impaired binding of stem cells to the endothelium via fucoidan
binding to P- and/or L-selectins, which play a central role in the inflammatory
response triggered by ischemia (P-selectin is upregulated on endothelial cells
and platelets during ischemic disorders) [15, 107], and, most likely, (ii) disrup-
tion of the SDF-1 gradient between blood and bone marrow or ischemic tissue.
is would modify the chemokine gradient that sustains the homing of stem
cells/leukocytes to ischemic sites and promotes their mobilization. In addition to
the contribution of EPCs to new vessel formation, enhanced SDF-1 expression,
which can activate EPCs, may also contribute to revascularization of ischemic
tissues [64]. is would also explain the impact of fucoidan on intimal growth
9.4 Biological Properties 287
Vasculogenesis
Angiogenesis
Bone marrow
sinusoid
(d)
(d)
(c)
(a)
(a)
FUC
FUC
FUC
FUC
FUC
FUC
FUC
FUC
FUC FUC
FUC
(b)
(e)
VEGF secretion
Ischemic
tissue
Laminin,
collagen,
etc.
SDF-1 EPC
Activated EPC
Laminin, collagen,
etc.
Fucoidan
Cationic proteins
Stromal cells
MMP9,
IL-8, etc.
Bone marrow
Blood vessel
Thrombin
TFPI
Fibrin
clot
PAI - 1
tPA
Figure 9.8 Proposed mechanisms involved in beneficial effects of fucoidan in peripheral
ischemia. (a) Fucoidan can bind to, potentiate, and inactivate cationic proteins such as
adherent proteins expressed in extracellular matrix (ECM) and bone marrow, enzymes, growth
factors, cytokines, and so on. (b) Fucoidan can prevent thrombosis by catalyzing thrombin
inhibition, promoting fibrinolysis, and stimulating the release of tissue factor pathway
inhibitor (TFPI). (c) Fucoidan can promote EPC recruitment by displacing sequestered SDF-1
from HSPG. It can also compete with EPC for binding heparan sulfate proteoglycans (HSPGs) or
ECM proteins. (d) Fucoidan can bind to EPC and induce a proangiogenic phenotype to EPC by
enhancing their mobility. (e) Fucoidan enhances EPC chemotaxis toward VEGF, EPC
attachment to laminin, and growth factor-induced vascular tube formation, improving
revascularization of ischemic tissue.
after stenting in rabbits and after allogeneic vascular transplantation in mice [84,
98]. Fucoidan can also act on surrounding cells such as smooth muscle cells, neu-
trophils, and macrophages attracted by cytokines released from an ischemic site
[111].
During transmigration through the endothelium, progenitors traverse the
endothelial cell monolayer and the subendothelial basement membrane.
Consequently, transmigrating cells interact with endothelial cells, GAGs, and
EMC adhesion proteins. Integrin α6, a laminin receptor involved in stem cell
homing to bone marrow [109], is expressed by EPCs. Its expression is required
for stem cell migration, and its overexpression after fucoidan treatment might
facilitate their homing toward ischemic sites. Finally, adhesion receptors like
selectins are important for cell migration and extravasation to ischemic sites.
During mobilization, traffic between bone marrow and blood is bidirectional.
According to Frenette and Weiss [58], blockade of endothelial selectins may
prevent progenitors from reentering bone marrow.
288 9 Use of Marine Compounds to Treat Ischemic Diseases
9.5 Conclusion
Development of a therapy capable of preventing tissue necrosis and of favoring
revascularization is crucial for patients with critical ischemia [112]. Although
marine polysaccharides have numerous interesting effects on coagulation,
thrombosis, inflammation, and angiogenesis, their clinical development is
hindered by their heterogeneity. Fucoidan is systemically distributed after
ingestion or intravenous injection and is nontoxic in mice when injected at doses
exceeding 1 g kg−1(private communication). Oral ingestion of fucoidan at doses
of 1–3 g day−1for up to 3 months is nontoxic in healthy volunteers [99, 113].
Cardiovascular disease is the leading cause of death worldwide (17.3 million
deaths each year, representing 30% of global mortality), and new anticoagulants
are urgently needed. LMWF possess several important advantages as alter-
native anticoagulant and proangiogenic therapies. First and foremost, they
are devoid of significant pro-hemorrhagic effects. Second, because of their
non-mammalian origin, there is no risk of contamination with viruses or
unconventional pathogens such as the prion responsible for bovine spongiform
encephalopathy. ird, as by-products of alginate preparation for the food and
cosmetics industries, they represent a cheap and readily available source of
new biologically active molecules. A better understanding of the mechanisms
by which these polysaccharides promote EPC activation and recruitment to
ischemic sites is necessary for optimal development of a proangiogenic therapy
product.
References
1Kiuru, P., D’Auria, M.V., Muller, C.D. et al. (2014) Exploring marine
resources for bioactive compounds. Planta Med.,80 (14), 1234–1246.
2Penesyan, A., Kjelleberg, S., and Egan, S. (2010) Development of novel
drugs from marine surface associated microorganisms. Mar. Drugs,8(3),
438–459.
3Tseng, C.K. and Chang, C.F. (1984) Chinese seaweeds in herbal medicine.
Hydrobiologia,116 (1), 152–154.
4Guiry, M.D. (2016) e Seaweed Site: Information on Marine Algae, http://
www.seaweed.ie/nutrition/index.php (18 January 2018).
5Ruocco, N., Costantini, S., Guariniello, S. et al. (2016) Polysaccharides from
the marine environment with pharmacological, cosmeceutical and nutraceu-
tical potential. Molecules,21 (5), pii: E551.
6Zaporozhets, T. and Besednova, N. (2016) Prospects for the therapeutic
application of sulfated polysaccharides of brown algae in diseases of the
cardiovascular system: review. Pharm. Biol.,54 (12), 3126–3135.
7Guiry, M.D. (2016) e Seaweed Site: Information on Marine Algae, http://
www.seaweed.ie/nutrition/index.php (18 January 2018).
8Kylin, H. (1915) Untersuchungen über die Biochemie der Meeresalgen.
HoppeSeyler’s Z. Physiol. Chem.,94 (5-6), 337–429.
References 289
9Deniaud-Bouët, E., Kervarec, N., Michel, G. et al. (2014) Chemical and enzy-
matic fractionation of cell walls from Fucales: insights into the structure of
the extracellular matrix of brown algae. Ann. Bot.,114 (6), 1203–1216.
10 Li, B., Lu, F., Wei, X. et al. (2008) Fucoidan: structure and bioactivity.
Molecules,13 (8), 1671–1695.
11 Chevolot, L., Foucault, A., Chaubet, F. et al. (1999) Further data on the
structure of brown seaweed fucans: relationships with anticoagulant activity.
Carbohydr. Res.,319 (1–4), 154–165.
12 Ale, M.T., Mikkelsen, J.D., and Meyer, A.S. (2011) Important determinants
for fucoidan bioactivity: a critical review of structure–function relations
and extraction methods for fucose-containing sulfated polysaccharides from
brown seaweeds. Mar. Drugs,9(10), 2106–2130.
13 Boisson-Vidal, C., Zemani, F., Caligiuri, G. et al. (2007) Neoangiogenesis
induced by progenitor endothelial cells: effect of fucoidan from marine algae.
Cardiovasc. Hematol. Agents Med. Chem.,5(1), 67–77.
14 Bachelet,L.,Bertholon,I.,Lavigne,D.et al. (2009) Affinity of low molec-
ular weight fucoidan for P-selectin triggers its binding to activated human
platelets. Biochim. Biophys. Acta,1790 (2), 141–146.
15 Rouzet, F., Bachelet-Violette, L., Alsac, J.-M. et al. (2011) Radiolabeled
fucoidan as a p-selectin targeting agent for in vivo imaging of platelet-rich
thrombus and endothelial activation. J. Nucl. Med. Off. Publ. Soc. Nucl. Med.,
52 (9), 1433–1440.
16 Suzuki, M., Bachelet-Violette, L., Rouzet, F. et al. (2015) Ultrasmall super-
paramagnetic iron oxide nanoparticles coated with fucoidan for molecular
MRI of intraluminal thrombus. Nanomedicine,10 (1), 73–87.
17 Fitton, J.H., Stringer, D.N., and Karpiniec, S.S. (2015) erapies from
fucoidan: an update. Mar. Drugs,13 (9), 5920–5946.
18 Hwang, P.-A., Yan, M.-D., Lin, H.-T.V. et al. (2016) Toxicological evaluation
of low molecular weight fucoidan in vitro and in vivo.Mar. Drugs,14 (7),
121.
19 Norgren, L., Hiatt, W.R., Dormandy, J.A. et al. (2007) Inter-society consen-
sus for the management of peripheral arterial disease (TASC II). J. Vasc.
Surg.,45 (1), S5–S67.
20 Lau, J.F., Weinberg, M.D., and Olin, J.W. (2011) Peripheral artery disease.
Part 1: Clinical evaluation and noninvasive diagnosis. Nat. Rev. Cardiol.,8
(7), 405–418.
21 Raval, Z. and Losordo, D.W. (2013) Cell therapy of peripheral arterial
disease: from experimental findings to clinical trials. Circ. Res.,112 (9),
1288–1302.
22 Dua, A. and Lee, C.J. (2016) Epidemiology of peripheral arterial disease and
critical limb ischemia. Tech.Vasc.Interv.Radiol.,19 (2), 91–95.
23 L’Artériopathie oblitérante, https://www.fedecardio.org/Les-maladies-cardio-
vasculaires/Les-pathologies-cardio-vasculaires/larteriopathie-obliterante
(18 January 2018).
24 Esmon, C.T. (2009) Basic mechanisms and pathogenesis of venous thrombo-
sis. Blood Rev.,23 (5), 225–229.
290 9 Use of Marine Compounds to Treat Ischemic Diseases
25 Weinberg, M.D., Lau, J.F., Rosenfield, K., and Olin, J.W. et al. (2011) Periph-
eral artery disease. Part 2: Medical and endovascular treatment. Nat. Rev.
Cardiol.,8(8), 429–441.
26 Foley, T.R., Waldo, S.W., and Armstrong, E.J. (2016) Antithrombotic therapy
in peripheral artery disease. Vasc. Med. Lond . Engl.,21 (2), 156–169.
27 Martin, B.-J. and Anderson, T.J. (2009) Risk prediction in cardiovascular dis-
ease: the prognostic significance of endothelial dysfunction. Can. J. Cardiol.,
25 (Suppl A), 15A–20A.
28 Sharma, S., apa, R., Jeevanantham, V. et al. (2014) Comparison of lipid
management in patients with coronary versus peripheral arterial disease.
Am.J.Cardiol.,113 (8), 1320–1325.
29 Oduah, E.I., Linhardt, R.J., and Sharfstein, S.T. (2016) Heparin: past, present,
and future. Pharmaceuticals (Basel),9(3), 38.
30 Rosenberg, R.D. and Damus, P.S. (1973) Correlation between structure and
function of heparin. J. Biol. Chem.,248, 6490–6495.
31 Choay,J.,Petitou,M.,Lormeau,J.C.et al. (1983) Structure–activity relation-
ship in heparin: a synthetic pentasaccharide with high affinity for antithrom-
bin III and eliciting high anti-factor Xa activity. Biochem. Biophys. Res. Com-
mun.,116 (2), 492–499.
32 Klein, A.J. and Ross, C.B. (2016) Endovascular treatment of lower extremity
peripheral arterial disease. Trends Cardiovasc. Med.,26, 495–512.
33 Rooke, T.W., Hirsch, A.T., Misra, S. et al. (2012) 2011 ACCF/AHA focused
update of the guideline for the management of patients with peripheral
artery disease (updating the 2005 guideline): a report of the American Col-
lege of Cardiology Foundation/American Heart Association Task Force on
Practice Guidelines: developed in collaboration with the Society for Cardio-
vascular Angiography and Interventions, Society of Interventional Radiology,
Society for Vascular Medicine, and Society for Vascular Surgery. Catheter.
Cardiovasc. Interv. Off. J. Soc. Card. Angiogr. Interv.,79 (4), 501–531.
34 Collinson, D.J. and Donnelly, R. (2004) erapeutic angiogenesis in periph-
eral arterial disease: can biotechnology produce an effective collateral
circulation? Eur.J.Vasc.Endovasc.Surg.Off.J.Eur.Soc.Vasc.Surg.,28
(1), 9–23.
35 Rissanen, T.T. and Ylä-Herttuala, S. (2007) Current status of cardiovascular
gene therapy. Mol. er. J. Am. Soc. Gene er.,15 (7), 1233–1247.
36 Pasqualoni, E., Messas, E., Fiessinger, J.-N. et al. (2008) Treatment perspec-
tives for critical limb ischemia: gene and cell therapy. Presse Méd.,37 (6 Pt
2), 1039–1046.
37 Kibbe, M.R., Hirsch, A.T., Mendelsohn, F.O. et al. (2016) Safety and efficacy
of plasmid DNA expressing two isoforms of hepatocyte growth factor in
patients with critical limb ischemia. Gene er.,23 (4), 399.
38 Emmerich, J. (2005) Current state and perspective on medical treatment of
critical leg ischemia: gene and cell therapy. Int. J. Low Extrem. Wounds,4
(4), 234–241.
39 Cooke, J.P. and Losordo, D.W. (2015) Modulating the vascular response to
limb ischemia: angiogenic and cell therapies. Circ. Res.,116 (9), 1561–1578.
References 291
40 Shyu, K.-G., Chang, H., Wang, B.-W. et al. (2003) Intramuscular vascular
endothelial growth factor gene therapy in patients with chronic critical leg
ischemia. Am. J. Med.,114 (2), 85–92.
41 Baumgartner,I.,Chronos,N.,Comerota,A.et al. (2009) Local gene transfer
and expression following intramuscular administration of FGF-1 plasmid
DNA in patients with critical limb ischemia. Mol. er. J. Am. Soc. Gene
er.,17 (5), 914–921.
42 Lawall, H., Bramlage, P., and Amann, B. (2011) Treatment of peripheral
arterial disease using stem and progenitor cell therapy. J. Vasc. Surg.,53 (2),
445–453.
43 Liang, T.W., Jester, A., Motaganahalli, R.L. et al. (2016) Autologous bone
marrow mononuclear cell therapy for critical limb ischemia is effective and
durable. J. Vasc. Surg.,63 (6), 1541–1545.
44 Ai, M., Yan, C.-F., Xia, F.-C. et al. (2016) Safety and efficacy of cell-based
therapy on critical limb ischemia: a meta-analysis. Cytotherapy,18 (6),
712–724.
45 Carmeliet, P. (2003) Angiogenesis in health and disease. Nat. Med.,9(6),
653–660.
46 Asahara, T., Kawamoto, A., and Masuda, H. (2011) Concise review: circulat-
ing endothelial progenitor cells for vascular medicine. Stem Cells Dayt. Ohio,
29 (11), 1650–1655.
47 Watt, S.M., Athanassopoulos, A., Harris, A.L. et al. (2010) Human endothe-
lial stem/progenitor cells, angiogenic factors and vascular repair. J. R. Soc.
Interface R. Soc.,7(Suppl 6), S731–S751.
48 Asahara, T., Masuda, H., Takahashi, T. et al. (1999) Bone marrow origin of
endothelial progenitor cells responsible for postnatal vasculogenesis in physi-
ological and pathological neovascularization. Circ. Res.,85 (3), 221–228.
49 Ho, T.K., Abraham, D.J., Black, C.M. et al. (2006) Hypoxia-inducible factor 1
in lower limb ischemia. Vascular,14 (6), 321–327.
50 Ho, T.K., Rajkumar, V., Ponticos, M. et al. (2006) Increased endogenous
angiogenic response and hypoxia-inducible factor-1alpha in human critical
limb ischemia. J. Vasc. Surg.,43 (1), 125–133.
51 Badorff, C. and Dimmeler, S. (2006) Neovascularization and cardiac repair
by bone marrow-derived stem cells, in Stem Cells,Handbook of Exper-
imental Pharmacology,vol.174 (eds A.M. Wobus and K.R. Boheler),
Springer-Verlag, Berlin Heidelberg, pp. 283–298.
52 Critser, P.J. and Yoder, M.C. (2010) Endothelial colony-forming cell role in
neoangiogenesis and tissue repair. Curr. Opin. Organ Transplant.,15 (1),
68–72.
53 Grisar, J.C., Haddad, F., Gomari, F.A. et al. (2011) Endothelial progenitor
cells in cardiovascular disease and chronic inflammation: from biomarker to
therapeutic agent. Biomark. Med.,5(6), 731–744.
54 Asahara, T., Murohara, T., Sullivan, A. et al. (1997) Isolation of putative pro-
genitor endothelial cells for angiogenesis. Science,275 (5302), 964–967.
55 Pearson, J.D. (2009) Endothelial progenitor cells – hype or hope? J. romb.
Haemost.,7(2), 255–262.
292 9 Use of Marine Compounds to Treat Ischemic Diseases
56 Silvestre, J.-S., Smadja, D.M., and Lévy, B.I. (2013) Postischemic revascu-
larization: from cellular and molecular mechanisms to clinical applications.
Physiol. Rev.,93 (4), 1743–1802.
57 Chavakis, E., Urbich, C., and Dimmeler, S. (2008) Homing and engraftment
of progenitor cells: a prerequisite for cell therapy. J. Mol. Cell. Cardiol.,45
(4), 514–522.
58 Frenette, P.S. and Weiss, L. (2000) Sulfated glycans induce rapid hematopoi-
etic progenitor cell mobilization: evidence for selectin-dependent and
independent mechanisms. Blood,96 (7), 2460–2468.
59 Sweeney, E.A., Lortat-Jacob, H., Priestley, G.V. et al. (2002) Sulfated polysac-
charides increase plasma levels of SDF-1 in monkeys and mice: involvement
in mobilization of stem/progenitor cells. Blood,99 (1), 44–51.
60 Roux, N., Brakenhielm, E., Freguin-Bouillant, C. et al. (2012) Progenitor cell
mobilizing treatments prevent experimental transplant arteriosclerosis. J.
Surg. Res.,176 (2), 657–665.
61 Dimmeler, S. and Leri, A. (2008) Aging and disease as modifiers of efficacy
of cell therapy. Circ. Res.,102 (11), 1319–1330.
62 Oda, M., Toba, K., Kato, K. et al. (2012) Hypocellularity and insufficient
expression of angiogenic factors in implanted autologous bone marrow in
patients with chronic critical limb ischemia. Heart Vessels,27 (1), 38–45.
63 Bitterli, L., Afan, S., Bühler, S. et al. (2016) Endothelial progenitor cells as a
biological marker of peripheral artery disease. Vasc. Me d . Lond. Engl.,21 (1),
3–11.
64 Zemani, F., Silvestre, J.-S., Fauvel-Lafeve, F. et al. (2008) Ex vivo priming
of endothelial progenitor cells with SDF-1 before transplantation could
increase their proangiogenic potential. Arterioscler. romb. Vasc. Biol.,28
(4), 644–650.
65 Boisson-Vidal, C., Colliec-Jouault, S., Fischer, A.M. et al. (1991) Biological
activities of fucans extracted from brown seaweeds. Drugs Future,16 (6),
539–545.
66 Springer, G.F., Wurzel, H.A., Mcneal, G.M. et al. (1957) Isolation of anti-
coagulant fractions from crude fucoidin. Proc. Soc. Exp. Biol. Med.,94 (2),
404–409.
67 Colliec, S., Boisson-Vidal, C., and Jozefonvicz, J. (1994) A low molecular
weight fucoidan fraction from the brown seaweed Pelvetia canaliculata.
Phytochemistry,35 (3), 697–700.
68 Percival, E.G.V. and Ross, A.G. (1950) Fucoidin. Part I. e isolation and
purification of fucoidin from brown seaweeds. J. Chem. Soc., 717–720.
69 Pauw, N. and Persoone, G. (1988) Microalgae for aquaculture, in Micro-Algal
Biotechnology (eds M.A. Borowitzka and L.J. Borowitzka), Cambridge Uni-
versity Press, Cambridge, pp. 197–221.
70 Nardella, A., Chaubet, F., Boisson-Vidal, C. et al. (1996) Anticoagulant low
molecular weight fucans produced by radical process and ion exchange
chromatography of high molecular weight fucans extracted from the brown
seaweed Ascophyllum nodosum.Carbohydr. Res.,289, 201–208.
71 Berteau, O., McCort, I., Goasdoué, N. et al. (2002) Characterization of a new
alpha-L-fucosidase isolated from the marine mollusk Pecten maximus that
References 293
catalyzes the hydrolysis of alpha-L-fucose from algal fucoidan (Ascophyllum
nodosum). Glycobiology,12 (4), 273–282.
72 Boisson-Vidal, C., Chaubet, F., Chevolot, L. et al. (2000) Relationship
between antithrombotic activities of fucans and their structure. Drug Dev.
Res.,51, 216–224.
73 Kloareg, B. and Quatrano, R.S. (1988) Structure of the cells walls of
marine algae and ecophysiological functions of the matrix polysaccharides.
Oceanogr. Mar. Biol. Annu. Rev.,26, 259–315.
74 Ustyuzhanina, N.E., Bilan, M.I., Ushakova, N.A. et al. (2014) Fucoidans:
pro- or antiangiogenic agents? Glycobiology,24 (12), 1265–1274.
75 Chevolot, L., Mulloy, B., Ratiskol, J. et al. (2001) A disaccharide repeat
unit is the major structure in fucoidans from two species of brown algae.
Carbohydr. Res.,330 (4), 529–535.
76 McLellan, D.S. and Jurd, K.M. (1992) Anticoagulants from marine algae.
Blood Coagul. Fibrinolysis Int. J. Haemost. romb.,3(1), 69–77.
77 Casu, B., Oreste, P., Torri, G. et al. (1981) e structure of heparin
oligosaccharide fragments with high anti-(factor Xa) activity contain-
ing the minimal antithrombin III-binding sequence. Chemical and13C
nuclear-magnetic-resonance studies. Biochem. J.,197 (3), 599–609.
78 Lindahl, U., Bäckström, G., unberg, L. et al. (1980) Evidence for a
3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of
heparin. Proc.Natl.Acad.Sci.U.S.A.,77 (11), 6551–6555.
79 de Jesus Raposo, M.F., de Morais, A.M.B., and de Morais, R.M.S.C. (2015)
Marine polysaccharides from algae with potential biomedical applications.
Mar. Drugs,13 (5), 2967–3028.
80 Elsner, H., Broser, W., and Burgel, E. (1937) e presence of highly active
anticoagulants in red algae. Z. Physiol. Chem.,246, 244–249.
81 Cumashi, A., Ushakova, N.A., Preobrazhenskaya, M.E. et al. (2007) A com-
parative study of the anti-inflammatory, anticoagulant, antiangiogenic, and
antiadhesive activities of nine different fucoidans from brown seaweeds.
Glycobiology,17 (5), 541–552.
82 Church,F.C.,Meade,J.B.,Treanor,R.E.et al. (1989) Antithrombin activity
of fucoidan. e interaction of fucoidan with heparin cofactor II, antithrom-
bin III, and thrombin. J. Biol. Chem.,264 (6), 3618–3623.
83 Colliec, S., Fischer, A.M., Tapon-Bretaudiere, J. et al. (1991) Anticoagulant
properties of a fucoïdan fraction. romb. Res.,64 (2), 143–154.
84 Durand, E., Helley, D., Al Haj Zen, A. et al. (2008) Effect of low molecular
weight fucoidan and low molecular weight heparin in a rabbit model of
arterial thrombosis. J. Vasc. Res.,45 (6), 529–537.
85 Richard, B., Bouton, M.-C., Loyau, S. et al. (2006) Modulation of protease
nexin-1 activity by polysaccharides. romb. Haemost.,95 (2), 229–235.
86 Doctor, V.M., Hill, C., and Jackson, G.J. (1995) Effect of fucoidan during
activation of human plasminogen. romb. Res.,79 (3), 237–247.
87 Chabut, D., Fischer, A.M., Helley, D. et al. (2004) Low molecular weight
fucoidan promotes FGF-2-induced vascular tube formation by human
endothelial cells, with decreased PAI-1 release and ICAM-1 downregulation.
romb. Res.,113 (1), 93–95.
294 9 Use of Marine Compounds to Treat Ischemic Diseases
88 Matou, S., Helley, D., Chabut, D. et al. (2002) Effect of fucoidan on fibrob-
last growth factor-2-induced angiogenesis in vitro.romb. Res.,106 (4–5),
213–221.
89 Zemani, F., Benisvy, D., Galy-Fauroux, I. et al. (2005) Low-molecular-weight
fucoidan enhances the proangiogenic phenotype of endothelial progenitor
cells. Biochem. Pharmacol.,70 (8), 1167–1175.
90 Soeda, S., Kozako, T., Iwata, K. et al. (2000) Oversulfated fucoidan inhibits
the basic fibroblast growth factor-induced tube formation by human umbili-
cal vein endothelial cells: its possible mechanism of action. Biochim. Biophys.
Acta,1497 (1), 127–134.
91 Sarlon, G., Zemani, F., David, L. et al. (2012) erapeutic effect of
fucoidan-stimulated endothelial colony-forming cells in peripheral ischemia.
J. romb. Haemost.,10 (1), 38–48.
92 Luyt,C.-E.,Meddahi-Pellé,A.,Ho-Tin-Noe,B.et al. (2003)
Low-molecular-weight fucoidan promotes therapeutic revascularization
in a rat model of critical hindlimb ischemia. J. Pharmacol. Exp. er.,305
(1), 24–30.
93 Omata, M., Matsui, N., Inomata, N. et al. (1997) Protective effects of
polysaccharide fucoidin on myocardial ischemia-reperfusion injury in rats. J.
Cardiovasc. Pharmacol.,30 (6), 717–724.
94 Logeart, D., Prigent-Richard, S., Boisson-Vidal, C. et al. (1997) Fucans,
sulfated polysaccharides extracted from brown seaweeds, inhibit vascular
smooth muscle cell proliferation. II. Degradation and molecular weight
effect. Eur. J. Cell Biol.,74 (4), 385–390.
95 Patel, M.K., Mulloy, B., Gallagher, K.L. et al. (2002) e antimitogenic action
of the sulphated polysaccharide fucoidan differs from heparin in human vas-
cular smooth muscle cells. romb. Haemost.,87 (1), 149–154.
96 Deux, J.-F., Meddahi-Pellé, A., Le Blanche, A.F. et al. (2002) Low molec-
ular weight fucoidan prevents neointimal hyperplasia in rabbit iliac
artery in-stent restenosis model. Arterioscler. romb. Vasc. Biol.,22 (10),
1604–1609.
97 Hlawaty, H., Suffee, N., Sutton, A. et al. (2011) Low molecular weight
fucoidan prevents intimal hyperplasia in rat injured thoracic aorta through
the modulation of matrix metalloproteinase-2 expression. Biochem. Pharma-
col.,81 (2), 233–243.
98 Freguin-Bouilland, C., Alkhatib, B., David, N. et al. (2011) Syngeneic bone
marrow cell therapy prevents intimal proliferation in allogeneic vascular
transplantation. J. Surg. Res.,168 (1), 143–148.
99 Irhimeh, M.R., Fitton, J.H., and Lowenthal, R.M. (2007) Fucoidan ingestion
increases the expression of CXCR4 on human CD34+cells. Exp. Hematol.,
35 (6), 989–994.
100 Blondin, C., Fischer, E., Boisson-Vidal, C. et al. (1994) Inhibition of com-
plement activation by natural sulfated polysaccharides (fucans) from brown
seaweed. Mol. Immunol.,31 (4), 247–253.
101 Alkhatib, B., Freguin-Bouilland, C., Lallemand, F. et al. (2006) Low molecu-
lar weight fucan prevents transplant coronaropathy in rat cardiac allograft
model. Transpl. Immunol.,16 (1), 14–19.
References 295
102 Pomin, V.H. and Mourão, P.A.S. (2008) Structure, biology, evolution, and
medical importance of sulfated fucans and galactans. Glycobiology,18 (12),
1016–1027.
103 Fonseca, R.J.C., Oliveira, S.-N.M.C.G., Melo, F.R. et al. (2008) Slight dif-
ferences in sulfation of algal galactans account for differences in their
anticoagulant and venous antithrombotic activities. romb. Haemost.,
99 (3), 539–545.
104 Boisson-Vidal, C., Haroun, F., Ellouali, M. et al. (1995) Biological activities of
polysaccharides from marine algae. Drugs Future,20 (12), 1237–1249.
105 Teruya, T., Tatemoto, H., Konishi, T. et al. (2009) Structural characteristics
and in vitro macrophage activation of acetyl fucoidan from Cladosiphon
okamuranus. Glycoconj. J.,26 (8), 1019–1028.
106 Hiramatsu, Y., Tsujishita, H., and Kondo, H. (1996) Studies on selectin
blocker. 3. Investigation of the carbohydrate ligand sialyl Lewis X recogni-
tion site of P-selectin. J. Med. Chem.,39 (23), 4547–4553.
107 Bouvard, C., Galy-Fauroux, I., Grelac, F. et al. (2015) Low-molecular-weight
fucoidan induces endothelial cell migration via the PI3K/AKT pathway and
modulates the transcription of genes involved in angiogenesis. Mar. Drugs,
13 (12), 7446–7462.
108 Bouvard, C., Gafsou, B., Dizier, B. et al. (2010) α6-integrin subunit plays a
major role in the proangiogenic properties of endothelial progenitor cells.
Arterioscler. romb. Vasc. Biol.,30 (8), 1569–1575.
109 Qian, H., Tryggvason, K., Jacobsen, S.E. et al. (2006) Contribution of α6
integrins to hematopoietic stem and progenitor cell homing to bone marrow
and collaboration with α4integrins.Blood,107 (9), 3503–3510.
110 Pomin, V.H. (2012) Fucanomics and galactanomics: current status in drug
discovery, mechanisms of action and role of the well-defined structures.
Biochim. Biophys. Acta,1820 (12), 1971–1979.
111 Sapharikas, E., Lokajczyk, A., Fischer, A.-M. et al. (2015) Fucoidan stim-
ulates monocyte migration via ERK/p38 signaling pathways and MMP9
secretion. Mar. Drugs,13 (7), 4156–4170.
112 Pineda, J.R.E.T., Kim, E.S.H., and Osinbowale, O.O. (2015) Impact of phar-
macologic interventions on peripheral artery disease. Prog. Cardiovasc. Dis.,
57 (5), 510–520.
113 Myers, S.P., O’Connor, J., Fitton, J.H. et al. (2010) A combined phase I and
II open label study on the effects of a seaweed extract nutrient complex on
osteoarthritis. Biol. Targets er.,4, 33–44.
296 9 Use of Marine Compounds to Treat Ischemic Diseases
About the Author
Catherine Boisson-Vidal obtained her Ph.D. degree from Paris Nord
University (France), where Dr. Boisson-Vidal studied the isolation, struc-
tural elucidation, and structure–activity relationships of biologically active
marine fucoidans. She undertook postdoctoral research in biomaterials at
McMaster University (Hamilton, Canada) and took a lectureship at Paris
Nord (1986–2003) and Paris Descartes University, where she is currently a
CNRS research director and deputy director of Inserm research unit 1140.
Over the period 1989–2000, Dr. Boisson-Vidal’s main research focus was on
the development methods to prepare reproducible antithrombotic fractions
of low molecular weight fucoidan and their structure–function relationship.
She is currently developing new therapeutic approaches based on stem cell
therapy, notably by attempting to enhance the proangiogenic potential of
adult stem cells through their incorporation into sites of neovascularization,
in both physiological and pathophysiological conditions. Her main focus is
on the proangiogenic activity of fucoidan and its uses in vascular biotherapy
and tissue regeneration.
