Background to the research
Marfan’s syndrome (MFS) is a severe, systemic disorder of
connective tissue formation and can lead to aortic aneurysms,
ocular lens dislocation, emphysema, bone overgrowth and severe
periodontal disease. MFS has an estimated prevalence of 1 in
5,000-10,000 individuals. Various mouse models of MFS have been
established via gene targeting or missense mutations, with germline
mutations in fibrillin-1 leading to progressive connective tissue
destruction due to fibrillin-1 fragmentation in association with an
insufficiency of fibrillin-1 microfibril formation. Hence, it is
largely accepted that MFS is caused by insufficient fibrillin-1
microfibril formation in various connective tissues.
A variety of MFS therapies have been developed, including surgical
therapy for aortic root aneurysms that are life-threatening,
traditional medical therapies such as β-adrenergic receptor
blockade for slow aortic growth and to decrease the risk of aortic
dissection, and novel approaches based on new insights such as the
pathogenesis of insufficient fibrillin-1 microfibril formation and
deregulation of Transforming Growth Factor-type beta (TGF-β)
activation. It has been demonstrated also that deregulation of
(TGF-β) activation contributes to MFS pathogenesis and that
matrix sequestration of TGF-β is critical for the regulated
activation and signaling of the extracellular fibrillin-1
microfibrils of connective tissues. Importantly, systemic
antagonism of TGF-β signaling through the administration of a
TGF-β neutralizing antibody or losartan, an angiotensin II
type 1 receptor blocker, has been shown to have a beneficial effect
on alveolar septation and muscle hypoplasia. These observations
provide a proof-of-principle for the use of TGF-β antagonism
is a general therapeutic strategy for MFS and other disorders of
the TGF-β signaling network. However, another potential
therapeutic strategy which remains to be investigated is the
reconstruction of the microfibril in connective tissues through the
expression or administration of a microfibril-associated molecule
that regulates or stabilizes fibrillin-1 microfibril formation. To
investigate this concept, it will be necessary to identify
molecular mechanisms of microfibril formation and an appropriate
fibrillin-1 microfibril associated molecule (Fig .1).
Fig. 1. Schematic representation of the MFS and ECM reinforcement
therapy as a novel therapeutic strategy for the treatment of
MFS.
Left panel : Fibrillin-1 comprises insoluble extracellular matrix
components in connective tissue microfibrils and provides limited
elasticity to tissues through fibrillin-1 microfibril formation.
Missense mutations of fibrillin-1 leading to progressive connective
tissue destruction due to fibrillin-1 fragmentation in association
with an insufficiency of fibrillin-1 microfibril formation.
ADAMTSL6β is essential for fibrillin-1 microfibril formation
and suggest a novel therapeutic approach to the treatment of MFS
through the promotion of ADAMTSL6β-mediated fibrillin-1
microfibril assembly.
Right Panel : A variety of MFS therapies have been developed,
including surgical therapy for aortic root aneurysms that are
life-threatening, traditional medical therapies such as
β-adrenergic receptor blockade for slow aortic growth and to
decrease the risk of aortic dissection, and novel approaches based
on new insights such as the deregulation of TGF-β activation.
ECM reinforcement therapy which induces restoration of properly
formed microfibrils by ADAMTSL6β is essential not only for
improvement of the predominant symptoms of MFS, but also for the
suppression of excessive TGF-β signaling induced by
microfibril disassembly.
Outline of the research outcome
1. Identification of ADAMTSL6β which induces microfibril
assembly.
The novel ADAMTSL family molecules ADAMTSL6α and 6β were
recently identified by in silico screening for novel ECM proteins
produced from a mouse full-length cDNA database (FANTOM). These
proteins are localized in connective tissues, including the skin,
aorta and perichondrocytes. Among the ADAMTSL6 family,
ADAMTSL6β has been shown to associate with fibrillin-1
microfibrils through its direct interaction with the N-terminal
region of fibrillin-1, and thereby promotes fibrillin-1 matrix
assembly in vitro and in vivo. These findings suggest a potential
clinical application of ADAMTSL6β as a novel MFS therapy by
promoting fibrillin-1 microfibril assembly and regulating
TGF-β activation.
To investigate whether ADAMTSL6β plays a critical role in
microfibril assembly in connective tissues, we generated
ADAMTSL6β transgenic mice (TSL6βTGmice) in which the
transgene is expressed in the whole body. Since ADAMTSL6β has
been shown to be expressed in the aorta and skin, we investigated
microfibril assembly of these tissues in the TSL6βTGmice.
Immunohistochemical analysis revealed that ADAMTSL6β positive
microfibril assembly was barely detectable in WT mice but strongly
induced in the aorta of TSL6βTGmice (Fig. 2). Histological
analysis revealed that microfibrils are clearly increased in the
aorta and that microfibril assembly is also induced in the skin and
PDL of TSL6βTG. mice. This confirmed that ADAMTSL6β
induces fibrillin-1 microfibril assembly in connective tissue such
as the aorta, skin and PDL.
Fig. 2. Immunohistochemical analysis of TSL6β-TG mice.
Cryosections were prepared from the aortas (left), skin (middle) or
PDL (right) of wild type (upper panel) or TSL6βTG (lower
panel) littermates and subjected to double immunostaining with
antibodies against ADAMTSL6β (red) and fibrillin-1 (green).
ADAMTSL6β and fibrillin-1-positive microfibrils (green yellow)
was markedly increased in the aorta and skin of TSL6β TG mice
compared with WT mice. Bar=50 µm
2. ADAMTSL6. is involved in microfibril restration during PDL wound
healing.
In our current study, we report that ADAMSL6β is essential for
the development and regeneration of the connective tissue
periodontal ligament (PDL), a tooth-supporting tissue located
between the root and alveolar bone which is morphologically similar
to the ligament tissue that is capable of withstanding mechanical
force. To investigate whether ADAMSL6β contributes to
connective tissue formation, we first examined its expression
patterns during PDL formation stage after birth as a model of
connective tissue formation. In situ hybridization analysis
revealed that ADAMSL6β was strongly expressed in the PDL
forming stage of the DF however ADAMSL6β expression was
significantly downregulated in the adult PDL(Fig. 3A upper).
Immunohistochemical analysis further revealed that ADAMSL6β is
detectable in assembled microfibril-like structures during the PDL
forming stage of the DF, and in organized microfibrils in the adult
PDL (Fig. 3A lower). Because developmental processes involve
similar mechanisms to wound healing, we next determined whether
ADAMSL6β is involved in PDL microfibril assembly during wound
healing using a tooth replantation model. Histochemical analysis
revealed an injured PDL with an irregular architecture at 3 days
after replantation, although gradual although gradual healing then
occurred at between 7 and 14 days after replantation. During these
processes, adamtsl6β mRNA expression were found to be clearly
induced in the PDL at 3 to 7 days after replantation, but to
decrease again by 14 days after replantation (Fig. 3B). Similar to
these gene expression patterns, adamtsl6- and fibrillin-1-positive
microfibrillar-like structures resembling those seen in the DF
during the PDL forming stage were markedly increased in the damaged
PDL at 3 to 7days after replantation.
Fig. 3. ADAMSL6β is involved in restoration of microfibril in
PDL.
A) ADAMSL6β mRNA (arrows) by specific probes at the P1-late
bell stage of dental follicle formation in the tooth germ is
indicated by arrows (upper). Expression of ADAMSL6β
microfibrils was detected in dental follicle during PDL forming
stage is indicated by arrows (lower).
B) Histological analysis of injured PDL 3 days, 7 days and 14 days
after replantation of the tooth were analyzed by histological
analysis (upper). Histological analysis indicated that expression
of ADAMSL6β mRNA were markedly increased 3 days and 7 days
after injury (middle). Immunohistochemical analysis using
anti-ADAMSL6β (TSL6 : green) and anti-fibrillin-1 (Fbn-1: red)
antibodies indicated that expression of ADAMSL6β- and
fibrillin-1-positive microfibrils was markedly increased 3 days and
7 days after injury (Lower). AB:alveolar bone, AM:ameloblast,
PDL:periodontal ligament, D:dentin, P:pulp
3. The local administration of ADAMTSL6β improves wound
healing ability in a MFS model.
We next investigated whether ADAMTSL6β might be developed as a
novel therapeutic for MFS microfibril disorder. Collagen gel
containing recombinant ADAMSL6β was locally administrated into
an experimentally damaged PDL in MFS mice model (Fig. 4A, B left).
Histochemical analysis showed that a reorganization of microfibril
assembly and wound healing could be observed after 17 days of
incubation (Fig. 4B right). In contrast, the administration of
control collagen gel failed to induce PDL healing and microfibril
formation (Fig. 4B right ).
We next investigated whether ADAMTSL6β alleviates fibrillin-1
microfibril disorder in periodontal ligament cells obtained from
MFS patient (MHPDL), which shows a reduction in fibrillin-1
microfibril assembly (Fig. 4C left). We next investigated whether
recombinant ADAMSL6β improves the symptoms of MHPDL
microfibril disorder. We found that recombinant ADAMSL6β
induces microfibril assembly in MHPDL cells during three days
incubation in culture (Fig. 4C). These results illustrate that
reorganization of microfibrils by recombinant ADAMSL6β
improves structurally damaged microfibril in MFS.
Fig. 4 ADAMSL6β improves microfibril disorder in PDL from an
MFS model.
A) Schematic representation of the local administration of
recombinant ADAMSL6β into a PDL injury model
B) After injury of PDL by dislocation, collagen gel-containing
recombinant ADAMSL6β was then injected into the injured PDL
(left). Immunohistochemical analysis showed an improvement in
fibrillin-1 microfibril assembly (arrowheads) induced by the
injection of recombinant ADAMSL6β. WO:Without treatment of
ADAMSL6β.
C) Histological analysis of PDL cells obtained from MFS treated
with recombinant ADAMTSL6β. PDL cells obtained from MFS
patients showed microfibril insufficiency compared with PDL cells
obtained from healthy patient (left). Administration of
ADAMSL6βmarked improvement of microfibril assembly in PDL
cells obtained from MFS patients (right).
In conclusion, we provide evidence for the contributions of
ADAMTSL6β-mediated fibrillin-1 microfibril assembly to PDL
development, regeneration, and alleviation of MFS manifestations.
Wethereby introduce the concept that an ECM reinforcement therapy
such as ADAMTSL6β administration which induces microfibril
assembly, should be considered in the development of future
mechanism-based therapeutics for the improvement of connective
tissue disorders such as MFS. Our data suggest that the
reinforcement of fibrillin-1 assembly by ADAMTSL6β accelerates
the sequestration of newly synthesized TGF-β. It will also be
necessary to develop methodologies for the systemic administration
of ADAMTSL6β to induces microfibril assembly in connective
tissue for the treatment of life-threatening conditions such as
aortic aneurysm. Since elastolysis occurs continuously in aortic
aneurysms in MFS, chronic administration of ADAMTSL6βmay be
required for the stabilization of microfibrils to prevent
progressive tissue destruction. This approach will facilitate drug
discovery for treating MFS and related connective tissue
disorders.
Fig. 5 ECM reinforcement therapy as a novel therapeutic strategy of
MFS syndrome. ECM reinforcement therapy such as ADAMTSL6β
administration which induces microfibril assembly, should be
considered in the development of future mechanism-based
therapeutics for the improvement of connective tissue disorders
such as MFS.
|
