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Journal of Health Science, 57(6) 449–457 (2011)— Review —Role of Elastic Fibers on Cardiovascular Disease
Hiroshi Wachi∗
Department of Clinical Chemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2–4–41 Ebara, Shinagawa-ku,
Tokyo 142–8501, Japan(Received August 24, 2011)

An important factor in the transition from an open to a closed circulatory system was a change in vessel
wall that are dynamic structure composed of cells and extracellular matrix. The component of arterial wall invertebrates that accounts for these properties is the elastic fiber network organized by medial smooth muscle cells.
Elastin and elastin associated protein are synthesized and secreted by vascular smooth muscle cells and are themajor extracellular matrix component deposited in the vascular wall. Pathological states related to hypertension or
atherosclerosis is associated with vascular wall remodeling, which is deleterious for cardiovascular function. Elastic
fiber may be key factors in the pathophysiology of hypertensive or atherosclerotic vascular remodeling. The wellknown effects of cardiovascular disease on the deterioration and the promoted degradation of elastic fiber result toloss of arterial wall resilience. Recently, several studies have highlighted new roles for individual components ofelastic fiber and their degraded products. This review describes current knowledge regarding components of elastic
fibers and discusses relationship between their structural abnormalities and cardiovascular diseases.
Key words —— elastin, microfibrils, supravalvular aortic stenosis, elastic fiber assembly

INTRODUCTION
The extracellular matrix (ECM) plays a criticalrole in the development, growth and biomechanicalproperties of virtually every organ system. Elastin is
the dominant extracellular matrix protein depositedin the arterial wall and can contribute up to 50% of
its dry weight.1) The protein product of the elastin
gene is synthesized by vascular smooth muscle cells
and secreted as a tropoelastin monomer, that is soluble, non-glycosylated and highly hydrophobic. After posttranslational modifications, tropoelastin iscrosslinked and organized into elastin polymers thatform concentric rings of elastic lamellae around the
medial layer of arteries. In arteries, elastin dictates
tissue mechanics at low strains before stiffer collagen fibers are engaged.2) Elastin also confers elasticity, preventing dynamic tissue creep by stretching under load and recoiling to their original configurations after the load is released. In addition to
the mechanical responsiveness, elastin is a potent∗

To whom correspondence should be assessed: Departmentof Clinical Chemistry, Hoshi University School of Pharmacy
and Pharmaceutical Sciences, 2–4–41 Ebara, Shinagawa-ku,
Tokyo 142–8501, Japan. Tel. & Fax: +81-3-5498-5243; E-mailwchrs 1107@hoshi.ac.jp

autocrine regulator of vascular smooth muscle cells
activity and this regulation is important for preventing fibrocellular pathology. The present review focuses on role of elastic fibers that affect the vascularfunctions.

BIOLOGY OF ELASTIN
The heart and arteries are always active mechanically and seem to wear out more than any other system or organs. The potential energy accumulated in
the stretching of the vessel wall during contractionof the heart (systole) is dissipated in elastic recoil of
the wall during the period when the heart is inactive(diastole). This release of tension in the wall serves
as an auxiliary pump, forcing the blood forward during diastole. Thus, near the heart the flow of bloodis intermittent and it is elasticity of the wall. The
wall of the large conducting vessels makes it possible to have a continuous flow with an intermittent
pump. The normal human artery wall comprises
an endothelial cell with a few underling smoothmuscle cells (SMCs) which together make up tunica intima. The underlying tunica media contains
SMCs and their associated matrix only and is sepC 2011 The Pharmaceutical Society of Japan450

arated from the intima by the internal elastic lamina and from the surrounding adventitia which contains fibroblasts, small feeding blood vessels, andadventitial nerves, by the external elastic lamina. In
all blood vessels the components of connective tissue possess important structure and biomechanicalfunctions. The lamellae of elastin are important inproviding the elastomeric properties of the larger arteries and in the human aortic elastin represent 20–
50% of the total dry weight.
Elastin is the main component of elastic fiber,
which provides resilience and elasticity to many tissues such as skin, lungs, ligaments, and arterial
walls. Elastin fibers consist of two distinct components: elastin and microfibrils, whose components include fibrillin, microfibril-associated glycoprotein.3–5) Elastin is secreted from cells as a soluble protein of approximately 70-kDa that undergoes a complex assembly process requiring the coordinate expression of microfibrillar proteins. Major types of domains are found in tropoelastin: hydrophobic domains rich in glycine (Gly), valine(Val), proline (Pro) and alanine (Ala); hydrophilic
domains typical rich in lysine (Lys) and Ala involved in cross-linking.6) The C-terminus of tropoelastin is highly basic amino acid residue (RKRK)
and contains the only two cysteine (Cys) residues.7, 8) The human single elastin gene has been
localized to chromosome 7q11.1–21.1, spanning
45 kb.9) The human gene has 34 exons with an
intron: exon ratio of 20 : 1, indicating that relatively small exons are interspersed within large intron. Alu repeats are found in the human elastin
gene at a frequency four times higher than elsewhere in the genome raising the possibility inthis gene through recombination events in thegeneral population10, 11) and possibly contributing
to diseases such as supravalvular aortic stenosis(SVAS). Some variation exists in the presence ofexon homologues amongst species. For example,
bovine exons 34 and 35 are absent from the human gene, while the human gene has the introduction of the unusual hydrophilic-encoding exon
26A, not described in any species.10) Exon 36 ishighly conserved amongst species and codes for the
C-terminus in addition to a large 3-untranslated region suggesting the possibility of regulatory elements in this region. The decrease in elastin synthesis that occurs with age has been shown to result
partly from the destabilization of mRNA.
The primary transcript of tropoelastin undergoes extensive alternative splicing resulting in theVol. 57 (2011)translation of multiple heterogeneous protein isoforms.12) The alternative splicing of the elastin geneoccurs in a cassette-like fashion in which an exon iseither included or deleted in most cases. At least 11
human tropoelastin splice variants have been identified with six exons shown to be subject to alternative
splicing; exons 22, 23, 24, 26A, and 32.5, 10, 13) It has
been reported that elastin gene exon 26A is always
spliced out and that exon 32 is highly spliced out
in human skin fibroblasts.14, 15) Elastin gene exon
26A is a human-specific sequence and is rich in
charge and polar amino acids. On the other hand,elastin gene exon 32 is a hydrophobic domain andis known to be a frameshift mutated exon in patientswith cutis laxa (CL).16) Although the heterogeneityof tropoelastin may play an important role in physiological functions, little is known about the elastic fiber formation of exon 26A or exon 32 missing
tropoelastin.

ELASTIN SYNTHESIS17)
Elastin expression in cultured SMC was enhanced by potent inhibitors of SMC proliferation
including retinoic acid18) and heparin.19) While,
elastin expression was reduced by potent stimulators of SMC proliferation like EGF,20) angiotensinII21) and high K concentration.22) These results suggest that elastin expression and SMC proliferation
are coupled tightly and inversely: potent stimulatorsof cell proliferation may potentially inhibit elastin
expression and potent inhibitors for cell proliferation can stimulate elastin expression. Nitric oxide(NO), an endothelium-dependent relaxing factor, is
continuously produced NO synthetase in endothelium. Endothelial dysfunction causes decreased
NO release, resulting in a shared process involving the pathogenesis of atherosclerosis and neointima formation after angioplasty. We demonstrated
that NO inhibited SMC proliferation, while NO enhanced expression of elastin.23) On the other hand,endothelin-1, a potent vasoconstrictor, has been
isolated from cultured porcine aortic endothelial
cells. Endothelin-1 is known as a potent stimulator of cell proliferation and development of arterial
diseases such as atherosclerosis. We demonstrated
that endothelin-1 stimulated cell proliferation andendothelin-1 suppressed the elastin expression suchas EGF, angiotensin II. The inhibition of elastin
synthesis was completely prevented by an endothelin receptor antagonist. These results indicate that

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endothelin-1 can modulate the elastin mRNA expression via an endothelin A receptor and that theregulator for elastin expression may play an important role in elastogenesis and SMC proliferation
during the development of atherosclerosis.24)

ELASTIC FIBER FORMATION
Elastin is secreted as a soluble protein of approximately 70 kDa referred to as tropoelastin,which has alternating hydrophobic and crosslinking domains. In the extracellular space, secreted
tropoelastin molecules interact with 10–12 nm microfibrils, which are thought to facilitate crosslinking by aligning cross-linking domains between
individual tropoelastin molecules. Previous studies have demonstrated a direct interaction between
tropoelastin and microfibrils,25–27) and domainmapping studies have localized the microfibrilbinding domain to the carboxy-terminal regionof tropoelastin.28– 30) A member of the lysyl oxidase family of amine oxidases catalyzes the crosslinking reaction through oxidative deamination of
the epsilon-amino group of specific Lys side chainswith tropoelastin. There is substantial evidence
that the hydrophobic domains are necessary for the
self-aggregation of tropoelastin via coacervation,
which is thought to concentrate and align tropoelastin molecules for cross-linking.31) Hydrophobic
domains of tropoelastin are rich in amino acids
such as Gly, Pro, Val, and leucine (Leu), which
are present in a variety of tandem repeat sequences.
The tandem repeats; i.e., VGVAPG, GGLG(V/A)
that resemble sequences found in other proteins toaggregate via β-sheet/β-turn structures,32, 33) contribute to the polymerization of elastin.28, 34) Recently, it has been reported that exon 1635) and
30,28) which encodes hydrophobic domain containing a tandem repeat sequence GGLG(V/A) is
an important sequence for the assembly process.
Our recently data showed that conditions favoring tropoelastin coacervation improved fibulin-5
binding.36) Fibrillin-1 has recently been reported
to promote coacervation.37) Because fibulin-5 interacts with both fibrillin-1 and tropoelastins,38, 39)a fibulin-5–fibrillin-1 complex might cooperatively
promote coacervation. Moreover, we reported thatwhole molecule of tropoelastin is necessary forthe formation of cross-linking amino acids such as
desmosine.40) Based on these observations, we hypothesize that conformational changes associatedwith coacervation of tropoelastin are necessary forfibulin-5 binding to tropoelastin. This hypothesis would predict that tropoelastin-fibulin-5 binding
and tropoelastin coacervation are coupled molecular
processes and that fibulin-5 binding facilitates thecoacervation of tropoelastin and thereby enhanceselastic fiber formation. This is exactly what our experiments demonstrated. Addition of fibulin-5 totropoelastin indeed lowered its coacervation temperature consistent with enhanced coacervation of
tropoelastin. It has been also reported that fibulin5 interacts with lysyl oxidase like 1.41) Taken together, molecular interaction between tropoelastin
and fibulin-5 may play a more positive role in thealignment and maturation of tropoelastin.

GENETIC DISORDER OF ELASTICFIBERS AND VASCULAR DISEASE
Elastogenesis is a very complex process involving multiple hierarchical steps. Therefore, mutations in the genes controlling elastic fiber deposition
might also participate in the development of cardiovascular diseases in general and hypertension inparticular. The knowledge of the genes controlling
elastogenesis has expanded in recent years, thanks
to the studies of human genetic disorders of the elastic fiber and recent advances in targeted deletionsof genes involved in elastogenesis in murine models. The fact that most of these mutations have been
linked to abnormal vascular structure and, in somecases, with the development of hypertension, haveraised the possibility to explore polymorphisms ofthese genes in relation to human essential hypertension.42–44) The elastin gene at least 3 clinical conditions have been linked to mutation or deletion of
the elastin gene: autosomal dominant CL, SVAS,
and Williams syndrome (WS). CL is primarily acutaneous condition,16, 45, 46) while WS and SVAS
are both disorders linked to alterations in the cardiovascular system.46) SVAS, an inherited obstructive arterial disease, may occur as an isolated condition47) or as part of the complex developmental
syndrome, WS.48) Isolated SVAS is caused by alarge spectrum of mutations including point mutations, translocations, and partial deletions within theelastin gene.49–51) Hypertension is present in a large
proportion of SVAS and WS patients usually fromearly age.52–54) Development of elastin-null (ELN−/−)55) and hemizygous (ELN +/−) mice44) has reinforced the possible link between defective elastin

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synthesis and hypertension. Elastin-null mice dieof obstructive arterial disease due to SMC proliferation and reorganization in the absence of endothelial damage, thrombosis, or inflammation.55) The
arteries of ELN +/− mice exhibited several similarities with the human syndromes linked to elastin
gene defects, such as thinner lamellae and increased
number of lamellar units and are hypertensive froman early age.44) When subjected to high pressures,large vessels from ELN +/− mice are stiffer thanthose from control animals, similar to the alterations
observed in human essential hypertension.56, 57) The
vascular alterations in resistance arteries from ELN+/− mice have not been explored. The role ofelastin in the maintenance of resistance artery structure and mechanics that we have recently reported58) suggests the possibility that small artery narrowing might also contribute to the development ofhypertension in these mice.
Mutations in the genes coding for the microfibrillar proteins, fibrillin-1 and fibrillin-2 have been
linked to Marfan syndrome.59–61) The major sourceof morbidity and mortality in this syndrome are thecardiovascular abnormalities involving aortic dilatation and dissection, which indicates the importanceof fibrillins to vessel wall structure. There are some
reports showing an association between aneurismal
disease, systolic blood pressure, and polymorphic
variation in the fibrillin-1 gene.62, 63) A more recent report suggests an important role for fibrillin1 genotype in cardiovascular risk associated withlarge-artery stiffening and pulse pressure elevation
in individuals with coronary disease.64) Therefore,
the contribution of fibrillin-1 to the pathogenesis ofvascular defects in human hypertension is a possibility that deserves further investigation. Fibrillindeficient mouse models have also been developed.
These studies have demonstrated that the absence of
normal levels of fibrillin-165) and fibrillin-266) is incompatible with the proper function of mature elastic fibres. Fibrillin-1 deficient mice develop cardiovascular abnormalities similar to those found in the
Marfan syndrome, such as aneurisms.65) As in theelastin deficient murine model, SMC from fibrillin1 deficient mice also show a change from quiescent contractile state to synthetic proliferative phenotype. This signaling pathway may be the basis of
the fragility of the vascular wall that accompanies
Marfan syndrome, as this phenotypic change is associated with a subsequent release of matrix metalloproteinases (MMPs) that degrade the vascularwall.67)

Vol. 57 (2011)

The fibulins are a family of seven ECM proteins that contain EGF-like modules and a distinctive C-terminal domain.68–70) Fibulin-5 [also known
as developmental arteries and neural crest EGFlike (DANCE)] is an ECM protein expressed inlarge vessels during embryogenesis as well as inadult life. Inactivation of the fibulin-5 gene inmice results in a generalized elastinopathy affecting those organs with abundant elastic fibers, suchas skin, lung and blood vessels. The aorta offibulin-5 deficient mice presents abnormal morphology and altered elastic lamella.38, 71) In addition, it
has been reported that loss of compliance in fibulin5 −/− aorta is similar to those observed in aged human arteries.57, 72) However, the fact that abnormal
elastin organization was found soon after birth, suggest an underlying developmental defect rather thanan ageing process.38) An interesting finding is thatelastic fiber alterations were accompanied by a significant increase in systolic blood pressure and pulsepressure. These results indicate that the pulse dampening effect of the elastic arteries was disturbed owing to loss of elasticity in the aortic wall in mutant mice.38) Fibulin-5 also seem to be an important inhibitor of SMC proliferation and migration,
as demonstrated by the exaggerated remodeling of
the carotid artery upon vessel ligation in fibulin-5−/− mice compared to fibulin-5 +/+ controls.73)
Decreased lysyl oxidase (LOX) levels have been
detected in human hypertension.74) Therefore, it
could be argued that defective cross-linking ofelastin due to alteration in LOX might cause a decline in the resilience of arterial wall and participate in the development of hypertension. However,all 5 LOX isoenzymes are likely to use both elastin
and collagens as their substrates and a decrease in
LOX would also reduce tensile strength of the arterial wall. In fact, mice with inactivation of the LOX
gene (LOX −/−) develop aortic aneurisms and perinatal death.75) The lethality associated with the lackof only 1 isoenzyme, was unexpected. It is possible that LOX deficiency cannot be compensatedfor by other isoenzymes in terms of its contribution
to the development and maintenance of the structure and mechanical properties of the aortic wall.
These findings suggest that alterations in LOX activity may also play a critical role in human cardiovascular diseases, but its implication in the development of vascular structural abnormalities in hypertension remains to be determined.

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ABNORMAL ELASTIC FIBER AND
VASCULAR DISEASE
Atherosclerosis is associated with local accumulation of lipid and calcium in the atherosclerotic
plaque. It has suggested that one of the mechanism
involved in the deposition of lipids in the elastin
may be an interaction of the elastin protein withserum low density lipoprotein (LDL) resulting in atransfer of cholesterol ester to the elastin.76) Moreover, reduction of LDL is able to result from an
impairment in the binding site involved in the direct chemical binding of lipids to the elastin protein molecule.77) Previous studies indicated that local lipid deposition in the atherosclerotic plaque isdue to alteration of the elastin fraction, both quantitatively and qualitatively78) and close association
with elastin modulates the response of SMCs to hyperlipidemia and suggest a role for elastin in the formation of foam cells of smooth muscle origin fromatherosclerosis.79) These findings suggest that functional defense action of elastin against cholesterolsynthesis and SMC migration was disturbed by accumulation of lipid on elastic fiber and the deposition on elastic fiber induced change of elasticity or
physiological function in the arterial wall.
On the other hand, vascular calcification occurs
with increasing age and in association with a diverse range of diseases, including atherosclerosis,
diabetes, and uremia. Additionally, vascular calcification is now known to be present in 80% of
significant lesions and in at least 90% of patientswith coronary artery disease. The value of pulse
wave velocity (PWV), indicate of vascular elasticity, is positively associated with ageing, calcification and degree of atherosclerosis. Therefore, PWV
was used clinical diagnosis in Japan and these phenomena may be suggested that calcification or calcium deposition cause to inhibit elastin synthesis.
Our previous data showed that the decreased contents of the cross-links (desmosine) in the experimental atherosclerosis relates to down-regulationof lysyl oxidase and elastin maturation during arteriosclerosis with calcification.80)
In atherosclerosis, abnormal cholesterol andcalcium deposition in aortic elastin fiber was associated with alterations in the amino acid composition of elastin and cross-links. Recent studies
suggest that arterial and vascular calcification occurs through highly regulated molecular processes
characterized by expression of osteogenic proteinsand matrix-degrading proteinases. There are reports that proteolutic enzymes expressed by activated macrophages and myofibroblast-like cells
in atherosclerotic plaque progression and aortic
valve disease. During atherogenesis, macrophage
derived proteinase such as elastolytic cathepsins,
elastase, or matrix metalloproteinase (MMP-2, -9,
and -12) promote the degradation of elastic fiber,
which favors calcification through an increase ofelastin polarity that in turn enhances elastin affinity for calcium. Cardiovascular calcification commonly causes devastating complications, including
plaque rupture and aortic valve stenosis, which currently have no suitable therapeutic alternative beyond valve replacement.

FUTURE PERSPECTIVES
The present review has highlighted some common features between patients with cardiovasculardiseases and abnormal elastic fibers. Both show
similar alterations in vascular wall architecture andmechanical performance also seems to be a common characteristic. These findings, together withthe fact that intact elastic fibers are essential for thenormal functioning of vascular mechanics, suggest
the possibility that elastic fiber abnormalities during
the fetal and early postnatal life might play a pivotal
role in the development of cardiovascular diseases.
The most consistent and well-reported changes are
luminal enlargement with arterial wall thickening(remodeling) and a reduction of elastic properties(stiffening) at the level of large elastic arteries. The
understanding of elastogenesis and prevention ofelastic fiber degradation would be very importantfrom the therapeutic point of view for cardiovascular diseases. For example, the effect of current
antihypertensive treatments on elastic fiber deposition and metabolism needs attention. Furthermore,
knowledge gained on the association between elastic fibers and cardiovascular diseases would allow
for the development of new therapeutic strategies.
Finally, in the light of the influence that minerals, vitamins, and dietary supplements exert on elastogenesis, the role of diet during pregnancy and in early
infancy on elastic fiber deposition and vascular remodeling deserves to examined, especially since it
implies the possibility of prevention of cardiovascular diseases.

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