「様式３－B」

課題番号（36）

Roles of vasohibins - CCN2/CTGF interactions inangiogenesis and cartilage/bone-related tumors［１］組織（Research group）
代表者（Principal Investigator (PI)）：
KHATTAB，HANY Mohamed Hanafi（Okayama University Dental School,
Advanced Research Center for Oraland Craniofacial Sciences
対応者（Host researcher at IDAC）：
SATO, Yasufumi（東北大学加齢医学研究所）	  分担者（Co-investigator）：
TAKIGAWA，Masaharu(Advanced Research Center for Oraland Craniofacial Sciences, Okayama
University Dental School)
AOYAMA，Eriko(Advanced Research Center for Oraland Craniofacial Sciences, Okayama
University Dental School)
MURASE，Yurika(OkayamaUniversityGraduate
School of Medicene, Dentistry andPharmaceutical Sciences)
研 究 費 (Expenditure report of researchfunds)：Costs of Consumables 200,000YEN［２］研究経過（Research setup）
Angiogenesis is involved in various biologicalprocesses such as endochondral ossification andsolid tumor growth. It is regulated by the local
balance between angiogenesis stimulators andinhibitors.
Recently, Prof. Y. Sato isolated
vasohibin-1 (VASH1) as a negative feedback
regulator of angiogenesis produced by endothelial
cells (ECs) and subsequently vasohibin-2 (VASH2)
as a homologue of VASH1. VASH1 is expressed in
ECs to terminate angiogenesis, whereas VASH2 isexpressed in cells other than ECs to promoteangiogenesis in the mouse model of angiogenesis.
Thus, much attempt to clarify roles of VASHs intumor growth has been made so far but there has

been no report on role of VASHs in endochondral
ossification.
In the process of endochondral ossification,chondrocytes proliferate, differentiate, maturate
and become hypertrophic chondrocytes and thenblood vessels penetrate into cartilage, which is anavascular tissue, from bone rich in ECs. Prof. M.
Takigawa et al. isolated a gene named hcs24(hypertrophic chondrocyte specific gene 24), which
is predominantly expressed in hypertrophic
chondrocytes and also in actively proliferating andmigrating ECs. Its gene products Hcs24, recently
re-named CCN2 (also known as connective tissue
growth factor:CTGF) promotes proliferation anddifferentiation of chondrocytes, osteoblasts, andECs and induce angiogenesis in vivo, resulting
promotion of endochondral ossification. However,
the role of CCN2 in a variety of malignancies ishighly variable and controversial. This might bedue to unique moleculer characteristics of CCN2.
Because CCN2 binds to various growth factors,
extracellular matrix and cell surface receptors
through 4 modules in the molecule, CCN2 mayorchestrates extracelluar signaling by interaction
with the binding partners of which combinations(microenvironment) are different among cell types,
and differentiated states of the same cells, andVasohibins)in)tumors)&)angiogenesis)
Y.)Sato’s)group)

Angiogenesis)
regulators

CCN2/CTGF)in))
endochondral))ossiﬁcaBon))(M.)Takigawa’s)group)

Role)of)vasohibinsKCCN2/CTGF)interacBon)in))
endochodral)
ossiﬁcaBon)and)tumor)growth&)angiogenesis)disorders of this orchestration may lead tomalignancies.
Therefore, in this study, we aimed to reveal
the role of VASHs in cartilage/bone formation,
especially endochondral ossification, as well as
their malignant changes.
Specifically, weinvestigated the involvement of interaction with
CCN2 and VASHs, possible mutual regulation
between VASHs and CCN2 and its significance inendochondral ossification, co-expression of
VASH2 and CCN2 in growth plate chondrocytes.
To perform this project, we have discussed sooften by e-mails. Examples include on May 13
and 14, June 12, 26 and 29, July 24, November 11,24 and 27, December 3, 2015 and January 14, 25
and 28, February 2 and 5. Moreover, we
discussed our results in detail at the 11th
Vasohibin Meeting at LA FORET ZAO, Zao onJanuary 23-24, 2016.
［３］成果（Research outcomes）（３－１）研究成果（Results）
During this fiscal year, we have obtained thefollowing results.
1) Mouse chondroprogenitor ATDC5 cells werecultured in the chondrogenic differentiation
medium for 4 weeks and gene expression ofVASH1, 2, CCN2, VEGF, MMP13, Col2a1,aggrecan, ColXal, and alkaline phosphatase(ALP) were analyzed by RT-PCR. As a result,gene expression of CCN2 and VASH1
increased as gene expression of ColXal, ALP,
MMP13, which are makers of hypertrophic
chondrocytes, increased. Gene expression ofVEGF, another angiogenesis factor, increasedslightly later than that of CCN2 and VASH2.
2) Expression of VASH2 was high in the earlystage of differentiation of ATDC5 anddecreased gradually as the cells differentiated.
3) Human chondrocytic cell line HCS-2/8 express
collagen type II and aggrecan which are two
major makers of chondrocytes but does notdifferentiate to hypertrophyic chondrocytes.
Expression level of VASH1 in HCS-2/8 cells
was low. Taken together with the finding in 1),
the result suggests thatVASH1 isexpressed only in hypertrophic chondrocytes
during chondrocyte differentiation.

4) When ATDC5 cells at early hypertrophic stage
were treated with recombinant CCN2, mRNAlevel of VASH1 was decreased after 12 h.
5) Before investing interaction between CCN2
and vasohibins, we investigated interaction
between various angiogenic factors and CCN2,and found that FGF-1, FGF-2 and VEGF
bound to CCN2 with high affinities.
（ ３ － ２ ） 波 及 効 果 と 発 展 性 な ど （Futureperspectives）
There had been no report on relation between
vasohibins and cartilage metabolism or on
relation between vasohibins and CCN proteins.
This study showed at the first time that VASH1
is involved in chondrocyte hypertrophy and theinvolvement has something to do with expressionof CCN2. These novel findings are fruits from acollaboration between two different research
fields/groups, producing a chance of developmentof new research fileds, e.g., “Vasohibins incartilage metabolism” and “Interaction between
vasohibins and CCN proteins”.
［４］成果資料（List of Papers）
There has been no paper presenting the dataobtained from this collaboration yet.
Thefollowings are a related book and papers.
（１）	  Takigawa M.: CCN Proteins: Methods
and Protocols, Springer, in press.
（２）	  Aoyama E, Kubota S, Khattab HM,
Nishida T, Takigawa M.: CCN2
enhances RANKL-induced osteoclastdifferentiation via direct binding toRANK and OPG. Bone, 73:242-8, 2015.
（３）	  Khattab HM, Aoyama E, Kubota S,Takigawa M.: Physical interaction ofCCN2 with diverse growth factors
involved in chondrocyte differentiation
during endochondral ossification. J. Cell
Commun. Signal. 9(3):247-54, 2015.
Presentation at a meeting.
Murase Y, Aoyama E, Kattab H, Abd Elkader	 T, Suzuki Y, Sasaki A, Kubota S, Sato Y,Takigawa M: Role of vasohibins-CCN2/CTGF
interaction in endochondral ossification. The
11th Vasohibin Meeting, LA FORET ZAO, Zao,January 23-24, 2016