Project 5 Update

AIM 1: We are revising a manuscript (for Cell Reports) that shows how ADAMTS9 regulates the growth of arteries. Specifically, we show, using a new gene-trap mouse allele, that ADAMTS9 is essential for matrix remodeling in the umbilical cord that is a prerequisite for differentiation and proper orientation of vascular smooth muscle cells. Since null mice with a conventionally targeted Adamts9 allele die before cardiovascular development, we developed a floxed Adamts9 allele, with which conditional developmental and inducible post-natal Adamts9 deletion in VSMCs will be achieved. Combinatorial ADAMTS-deficient mice (i.e., Adamts1/Adamts4Adamts5 + Adamts9) will be made to determine their cooperative roles in the vascular wall.

In another manuscript we deleted ADAMTS9 conditionally in smooth muscle cells and found that it was required for proper development of myometrium. Lack of ADAMTS9 led to a profound alteration of uterine ECM and interfered with parturition. This work is being prepared for submission and it indicates that smooth muscle cells require ADAMTS9-mediated proteolysis of ECM for proper development.

We have found that combined deletion of ADAMTS1 and ADAMTS5 during mouse gestation leads to anomalies similar to human Tetralogy of Fallot and that this phenotype also involves reduced versican proteolysis.

AIM 2: In context of VSMCs, we found that arterial VSMCs in vivo express ADAMTS1, ADAMTS5 and ADAMTS9, whereas in vitro they primarily express ADAMTS9. RNAi for ADAMTS9 in cultured aortic and myometrial VSMCs led to reduced cell differentiation, cytoskeletal reorientation and affected cell migration.

In Foulcer et al JBC 2014, we reported development of an antibody that blocks versican proteolysis and showed that prevention of ADAMTS5 proteolysis of versican by an ADAMTS5-function blocking antibody led to myofibroblast conversion of dermal fibroblasts.

In ongoing work related to evaluating the role of versican, we recently identified that versican deficient mice form neither yolk sac blood islands nor a yolk sac vasculature, and we are investigating the role of the versican-HA complex in survival of the hemangioblast.

Publications supported by the PEG:

(1) Dubail J, Aramaki-Hattori N, Bader HL, Nelson CM, Katebi N, Matuska B, Olsen BR, Apte SS. A new Adamts9 conditional mouse allele identifies its non-redundant role in interdigital web regression. Genesis. 2014 52:702-12.

(2) Foulcer SJ, Nelson CM, Quintero MV, Kuberan B, Larkin J, Dours-Zimmermann MT, Zimmermann DR, Apte SS. Determinants of Versican-V1 Proteoglycan Processing by the Metalloproteinase ADAMTS5. J Biol Chem. 2014 Oct 3;289(40):27859-73.

(3) Foulcer SJ, Day AJ, Apte SS. Isolation and purification of versican and analysis of versican proteolysis. Methods Mol Biol. 2015;1229:587-604.

(4) Nandadasa S, Foulcer S, Apte SS. The multiple complex roles of versican and its proteolytic turnover by ADAMTS proteases during embryogenesis. Matrix Biol. 2014, 35:34-41

 

Trainees have benefitted greatly from the Skills Development and Resource cores of the PEG. Additionally, each trainee attended a National or International meeting where they presented their data from our supported PEG project.

Presentations as below:

2014 Gordon Research Conference on Proteoglycans

“Novel contexts for versican processing by ADAMTS proteases during morphogenesis”

July 11-16 2014, Proctor Academy, Andover, New Hampshire

2014 American Heart Association

“ADAMTS and ADAMTSLs in microfibril function and vascular disease”

November 15-19, 2014, Chicago, McCormick Place Convention Center

Poster presentations were made at the American Society for Matrix Biology, 2014 in Cleveland, OH

 

AIM 1: We are undertaking combined conditional deletion of ADAMTS9 in VSMCs together with deletion of ADAMTS1 and ADAMTS5 to investigate the impact on development of the aorta. We will complete the characterization of the Tetralogy of Fallot defect and determine whether a versican-hyaluronan complex is indeed crucial for haemangioblast development using mouse genetics and CRISPR-Cas9 deletion of versican. Based on the seminal findings made in Foulcer et al JBC, 2015, we are generating a mouse with a knock-in mutation of versican designed to eliminate versican cleavage by ADAMTS proteases. This new model will directly test the hypothesis that versican proteolysis is crucial for vascular development

AIM 2: We will investigate whether modulating versican proteolysis using anti-ADAMTS5, or the cleavage-blocking versican antibody described in Foulcer et al JBC, 2015 affects VSMCs. We will also treat these cells with the versican fragment versikine and determine the underlying mechanism. We will investigate further a new finding that versican is phosphorylated

 

1) Dubail J, Aramaki-Hattori N, Bader HL, Nelson CM, Katebi N, Matuska B, Olsen BR, Apte SS. A new Adamts9 conditional mouse allele identifies its non-redundant role in interdigital web regression. Genesis. 2014 52:702-12.

2) Foulcer SJ, Nelson CM, Quintero MV, Kuberan B, Larkin J, Dours-Zimmermann MT, Zimmermann DR, Apte SS. Determinants of Versican-V1 Proteoglycan Processing by the Metalloproteinase ADAMTS5. J Biol Chem. 2014 Oct 3;289(40):27859-73.

3) Foulcer SJ, Day AJ, Apte SS. Isolation and purification of versican and analysis of versican proteolysis. Methods Mol Biol. 2015;1229:587-604.

4) Nandadasa S, Foulcer S, Apte SS. The multiple complex roles of versican and its proteolytic turnover by ADAMTS proteases during embryogenesis. Matrix Biol. 2014, 35:34-41.

 

The Apte laboratory generated a new mouse floxed allele for conditional deletion of ADAMTS9. These mice were contributed to the Jackson Laboratories for distribution to interested investigators and have been issued the stock symbol B6.Cg-Adamts9tm1.1Apte/J (Stock# 26103, RRID JAX_026103). They can be found online at www.jax.org/strain/026103 and purchased at: www.jax.org/jax-mice-and-services/find-and-order-jax-mice.

 

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