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P7: Phenotypic switching of smooth muscle cells and immune responses in hyaluronan synthase 3-deficient mice

Current state of relevant research

In the neointima of atherosclerotic plaques hyaluronan (HA) synthesis occurs to build a highly dynamic provisional extracellular matrix (ECM) that is thought to confer important functions in phenotypic control of vascular smooth muscle cells (SMC) and immune cells. SMC are thought to be activated within the HA-rich matrix to proliferate and migrate. Recently, it was shown that HA can attract and trap monocytes in experimental models of colitis and in vitro evidence for a similar effect was proposed for HA-matrices elaborated by SMC under proinflammatory conditions. In addition in vitro studies support a role of HA in the immune synapse to activate T cells.

Preliminary work

In the preceding funding period HA-synthase (HAS) 3 has been identified to be the HAS isoenzyme that is increased early during atherosclerosis at the same time when HA accumulation and macrophage invasion occurs in ApoE deficient mice. Therefore it is hypothesized that HAS3 mediated increase of plaque HA might be key event that facilitates both, SMC proliferation and migration as well as inflammation. With respect to SMC-driven lesion formation the knock out of HAS3 inhibited neointimal hyperplasia after ligation of the carotid artery in mice [1]. During atherosclerosis in ApoE deficient mice the knock out of HAS3 was indeed effective to reduce atherosclerosis (Homann et al. in preparation).

To investigate the fate and the origin of SMC- and macrophage-like cells in atherosclerotic lesions, the group of Gary Owens has generated an SMC lineage tracing mouse. Specifically a SMC (Myh11)-specific tamoxifen-induced eYFP mouse was crossbred into the ApoE-/- background to investigate atherosclerosis. Using this SMC lineage tracing mouse it was shown for the first time that more than 80% of SMC-derived (YFP-positive) cells within advanced atherosclerotic lesions were negative for SMC markers. Of these nearly a third had activated multiple markers of macrophages meaning they have been mis-identified in virtually all previous studies in the field [2].

Mechanistically, two observations on the HAS3/ApoE-deficient mice are proposed for further study here: (i) effects on the SMC phenotype within the plaque as determined by flow cytometric and high resolution z-stack analyses of marker panels previously developed by the Owens lab and recently expanded based on in vivo genomic analysis of advanced BCA lesions +/- Klf4 KO [2] and (ii) decreased activation of T cells and decreased macrophage recruitment. Currently the HAS3/ApoE deficient mice have been crossed at UVA with the YFP-SMC reporter mouse developed by the Owens lab to be able to track SMC derived cells within the plaques of HAS3/ApoE deficient mice. Susanne Homann is performing first analysis of these mice during her stay at the UVA from November 2016 until April 2017. She is being trained primarily by the Owens lab senior Research Scientist and Lab Manager Olga Cherepanova and senior PhD student Alexandra Newman. Alexandra and another Owens lab graduate student Katie Owsiany, will play key roles in the studies outlined in the research plan below including both spending significant time performing studies in the Fischer lab in Düsseldorf.

Research objectives of the joint program

As detailed above we propose to continue to work with the Owens lab on the question if and how HAS3 mediated HA-synthesis affects the phenotypic switching of SMC during atheroprogression using the YFP-reporter mice and how the immune response might be guided by the HAS3 mediated effects on SMCs. Furthermore in the preceding funding period Katie Owsiany, an MD/PhD student from the Owens laboratory started to look into another potential modulator of both phenotypic switching and inflammation, lymphotoxin beta receptor (LTbR) signaling. Previously we showed that knock out of LTbR inhibits atherosclerosis by interference with monocyte recruitment. During her stay in Düsseldorf Katie Owsiany found out that LTbR has profound effects on the secretome of SMC that in turn affect macrophage activation. Furthermore LTb is known to regulate HA synthesis. The work is proposed to be continued in the second funding period and feeds well into the HAS3 project assuming that the response of SMC to HAS3 is modified by cytokine signaling. In addition, Maria Grandoch established through an exchange student Jack Hensien from UVA a collaboration with the McNamara laboratory addressing the regulation and function of HAS2 mediated HA-synthesis in adipose tissue by Id3. This work is proposed to be continued as a new project with Maria Grandoch as new PI.

  1. Kiene, L. S., Homann, S., Suvorava, T., Rabausch, B., Müller, J., Kojda, G., Kretschmer, I., Twarock, S., Dai, G., and Deenen, R. (2015) Deletion of hyaluronan synthase 3 inhibits neointimal hyperplasia. Arterioscler Thromb Vasc Biol 36, e9-e16
  2. Shankman, L. S., Gomez, D., Cherepanova, O. A., Salmon, M., Alencar, G. F., Haskins, R. M., Swiatlowska, P., Newman, A. A., Greene, E. S., and Straub, A. C. (2015) KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat Med 21, 628-637

Kiene, L. S., Homann, S., Suvorava, T., Rabausch, B., Muller, J., Kojda, G.,
Kretschmer, I., Twarock, S., Dai, G., Deenen, R., Hartwig, S., Lehr, S., Kohrer, K., Savani, R.
C., Grandoch, M., and Fischer, J. W. (2016) Deletion of Hyaluronan Synthase 3 Inhibits
Neointimal Hyperplasia in Mice. Arterioscler Thromb Vasc Biol 36, e9-16

Grandoch, M., Kohlmorgen, C., Melchior-Becker, A., Feldmann, K., Homann, S.,
Muller, J., Kiene, L. S., Zeng-Brouwers, J., Schmitz, F., Nagy, N., Polzin, A., Gowert, N. S.,
Elvers, M., Skroblin, P., Yin, X., Mayr, M., Schaefer, L., Tannock, L. R., and Fischer, J. W.
(2016) Loss of Biglycan Enhances Thrombin Generation in Apolipoprotein E-Deficient Mice:
Implications for Inflammation and Atherosclerosis. Arterioscler Thromb Vasc Biol 36, e41-50

Grandoch, M., Feldmann, K., Gothert, J. R., Dick, L. S., Homann, S., Klatt, C., Bayer,
J. K., Waldheim, J. N., Rabausch, B., Nagy, N., Oberhuber, A., Deenen, R., Kohrer, K., Lehr,
S., Homey, B., Pfeffer, K., and Fischer, J. W. (2015) Deficiency in lymphotoxin beta receptor
protects from atherosclerosis in apoE-deficient mice. Circ Res 116, e57-68

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