Research in our laboratory has lead to the development of a technique by which the peritoneal cavity is used as a bioreactor to grow tubes of myofibroblast-rich tissue for replacement of smooth muscle organs. In this procedure, implantation of foreign material (such as boiled egg white and sterile polypropylene tubing) in the peritoneal cavity induces an inflammatory response that elicits myeloid cell recruitment, and subsequent encapsulation of the foreign object. When tubes of these myofibroblast-rich tissue capsule are grafted into smooth-muscle rich organs such as an artery to replace excised segments, cells undergo (trans)differentiation into smooth muscle-like cells. Although the factors that induce myeloid cells to transdifferentiate to myofibroblasts in the peritoneal cavity are unknown, gene array analysis conducted in our laboratory identified the cytokines transforming growth factor beta (TGFβ) and leukemia inhibitory factor (LIF) as differentially expressed in the developing tissue capsule cells. Thus this thesis examines the potential role of both TGFβ and LIF in this process.
The first experimental chapter (Chapter 3) showed TGFβ receptor II (TGFβRII) expression detectable from day 3 of tissue capsule formation and remaining constant until day 21. Treatment of day 5 tissue capsule cells with TGFβ for 7 days induced a spindle-like morphology similar to myofibroblasts, while untreated cells were smaller and rounded. Treatment of tissue capsule cells in vitro with the TGFβ receptor kinase inhibitor LY364947 significantly reduced α-SM actin expression compared to the control group. In vivo studies, in which LY364947 was delivered by osmotic mini-pumps, demonstrated inhibition of tissue capsule development. In addition, in vitro and ex vivo siRNA knockdown of TGFβRII in day 5 tissue capsule cells resulted in reduced α-SM actin expression, thus confirming that TGFβ is a positive regulator of myofibroblast differentiation in the peritoneal foreign body response.
Chapter 4 showed that cells of the developing tissue capsule express receptors for LIF, with LIF receptor (LIFR)-α expression increased over the first 7 days after foreign body implantation, after which expression remained constant up to day 21. When day 5 tissue capsule cells were cultured with exogenous recombinant LIF for 7 days, their expression of the myofibroblast differentiation marker, α-smooth muscle (α-SM) actin, was not affected. Experiments in LIF knockout mice showed that the absence of this cytokine had no effect during any stages of tissue capsule development. Furthermore, in vivo and ex vivo knockdown of LIFR-α by siRNA had no significant effect on differentiation of early stage tissue capsule cells to myofibroblasts.
To further examine the in vitro and in vivo effects of LIF and TGFβ in this process, a lentiviral vector was designed to deliver short-hairpin RNA (shRNA) to target receptors for TGFβ and LIF. Firstly, lentiviral RNAi constructs (pHIVCS-siTGFβR-II and pHIVCS-siLIFR-α) were produced by cloning the siRNA inserts into the pHIV-CS backbone, a third-generation HIV-1 based lentiviral plasmid. Additionally, a pHIVCS-EGFP was constructed and used as a reporter gene. An optimal MOI was determined for day 5 tissue capsule cells after titration assays were performed with pHIVCS-EGFP. Transduction of cultured day 5 tissue capsule cells with pHIVCS-siTGFβR-II and pHIVCS-siLIFR-α reduced TGFβR-II and LIFR-α expression by approximately 50%; in vivo injection of the lentiviral constructs yielded similar gene silencing effects. Consistent with earlier findings, lentiviral-mediated knockdown of TGFβR-II, but not LIFR-α, inhibited the differentiation of early tissue capsule cells into myofibroblasts both in vitro and in vivo.
In summary, this thesis provides evidence that TGFβ, but not LIF, plays a role in the differentiation of myofibroblasts within the peritoneal-derived tissue capsule, with TGFβ inducing myofibroblast differentiation. It suggests that strategies to modulate expression of TGFβ may be applied to regulate myofibroblast growth and differentiation, not only for tissue engineering purposes but also for management of pathological conditions such as hypertrophic scarring and fibrosis.