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  • inhibitor of apoptosis TPCA reversibly binds to IKK and prev

    2018-10-20

    TPCA-1 reversibly binds to IKKβ and prevents it from phosphorylating IκBα for degradation and thereby blocks nuclear translocation of NF-κB (Kondo et al., 2008). Our results demonstrate that TPCA-1 may inhibit the stimulatory action of TNF-α on NF-κB. TPCA-1 partially inhibits secretion of constitutively secreted pro-inflammatory factors such as IL-6 and MCP-1, which suggests that constitutive secretion is not entirely dependent on NF-κB activation. The reduction of RANTES, GRO, and GCP-2 to basal levels may indicate that NF-κB activation mediates the secretion of these cytokines upon TNF-α treatment as TPCA-1 abrogates NF-κB activation. Our cytokine array results confirm that TPCA-1 abrogates the effect of TNF-α on production of pro-inflammatory cytokines by hMSCs. In addition, we demonstrated that TPCA-1 treatment did not inhibit secretion of beneficial paracrine factors such as growth factors, cytokines, and anti-inflammatory mediator TSG-6, which when secreted from lung embolized hMSCs attenuate MI in mice (Lee et al., 2009). Hence, angiogenic and TSG-6-mediated anti-inflammatory properties of hMSCs may not be compromised upon TPCA-1 treatment. In addition, continuous intracellular TPCA exposure to hMSCs might yield sustained binding to IKKβ, resulting in the inhibition of NF-κB and overcome reversible inhibition of IKKβ. TPCA-1 has non-specific effects of IKKβ inhibition on many cell types with potential undesirable side effects. In addition, MSCs demonstrate a “hit and run effect” of immune modulation (Levy et al., 2013) and are cleared from the body within a week post-transplantation. Hence it is desirable to intracellularly deliver TPCA-1 into hMSCs at a controlled rate for transient modulation of the pro-inflammatory secretome. In this study, we developed an engineering platform to intracellularly deliver TPCA-1 to hMSCs. μPs of 1–2 μm were internalized following anionic surface modification. By choosing PLGA polymer with a lactic inhibitor of apoptosis to glycolic acid ratio of 50:50 and low molecular weight of 10 kDa, we tailored the release kinetics of TPCA-1 to achieve a concentration to inhibit IKK2. Upon preconditioning of hMSCs with soluble TPCA prior to inhibitor of apoptosis stimulation with TNF-α, the hMSC secretome was similar to TNF-α stimulation alone (TNF-hMSC) suggesting that IKKβ inhibition may require continued presence of TPCA to prevent NF-κB activation. The sustained intracellular release of TPCA-1 enabled prolonged inhibition of IKKβ and competitively inhibited TNF-α from activating NF-κB mediated pro-inflammatory cytokine secretion. NF-κB signaling in cells is oscillatory in nature, and gene expression seems to depend on its dynamics (Nelson et al., 2004). Hence, under continuous TNF-α stimulus, NF-κB activation might be sustained. However, the effect of intracellular TPCA inhibition in microparticle-engineered hMSCs was maintained after multiple rounds of TNF-α stimulation suggesting that microparticle-engineered hMSCs are able to control the pro-inflammatory phenotype under sustained inflammatory stimulus. To demonstrate the functional relevance of microparticle-mediated hMSC secretome regulation, we focused on cardiac fibrosis. IL-6 (Ma et al., 2012), MCP-1, and other pro-inflammatory mediators augment monocyte migration (Lee et al., 2010) and induce differentiation of CF to CMF, which are the primary scar-forming cells post-MI. Expectedly, we found that while the elevated levels of pro-inflammatory mediators in CM from preconditioned hMSCs are ineffective in inhibiting monocyte migration, CM from TNF + TPCAμP-hMSCs effectively inhibited transwell migration of THP-1 cells by at least 2- to 3-fold, corresponding to reduced levels of pro-inflammatory mediators as seen by our secretome analysis. Control-hMSC CM also induced significant differentiation of CF to CMF demonstrating the presence of pro-inflammatory mediators secreted by hMSCs as well as those likely to be present in the culture serum. CM from control-hMSCs and μP-hMSCs have lower levels of pro-inflammatory factors (compared with TNF-α-stimulated hMSCs) yet the number of α-SMA+ CMF remained unchanged when CF were treated with these CM, suggesting that the secretome may lack anti-inflammatory modulators and growth factors. In addition, treatment with TPCA-1 alone did not cause a reduction in the number of α-SMA+ CMF, suggesting that an hMSC-mediated paracrine function is essential. While TPCA-1 pretreatment of hMSCs before TNF-α stimulation showed a significant reduction in CMF numbers, this was greatly enhanced when TPCA-1 was available intracellularly via the microparticles. CM from TNF + TPCAμP-hMSC likely prevented differentiation of CF to CMF due to the continued intracellular inhibition of IKK-mediated NF-κB activation, thus preventing the release of an hMSC pro-inflammatory secretome. Surprisingly, the CMF number was reduced by over 2-fold, suggesting that TPCA-1 may activate hMSC pathways that revert the CMF phenotype. We cannot rule out the possibility that other contributors in the hMSC secretome that were not profiled might also be contributing, and their action is facilitated by intracellular TPCA-1. For instance, in an in vitro 3D model of cardiac fibrosis under hypoxic conditions, reversal of CMF to the CF phenotype was shown to be due to reduced MSC TGF-β levels (Galie and Stegemann, 2014). Our assay revealed a similar trend in terms of collagen production from CF. CF treated with CM from control-hMSCs, or TPCApre + TNF-hMSCs or μP-hMSCs secreted elevated levels of collagen into the media suggesting that only inhibited levels of pro-inflammatory mediators could not be implicated in the reduction in collagen secretion. The reduced number of α-SMA+ CMF in CM from TNF + TPCAμP-hMSCs possibly contributed to the lower secretion level of collagen in the media (Figure 5B). Upon de-differentiation or lowered α-SMA expression, it is possible that CMFs lose collagen secretion ability. In regions of MI, CF switch to the myofibroblast phenotype due to stress from the infarct scar (Tomasek et al., 2002). High expression of α-SMA typical in such CMF (Teunissen et al., 2007) has been implicated for remodeling due to their high contractility (Santiago et al., 2010). In addition, the collagen secretion capacity of CMF is very high (Petrov et al., 2002). Overall, attenuation in the number of collagen-secreting α-SMA+ CMF could be beneficial in preventing pathological remodeling or irreversible scar formation and allowing cardiac regeneration.