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Background— Cell fate is fluid and may be altered experimentally by the forced expression of master regulators mediating cell lineage. Such reprogramming has been achieved with the use of viral vectors encoding transcription factors. We recently discovered that the viral vectors are more than passive vehicles for transcription factors because they participate actively in the process of nuclear reprogramming to pluripotency by increasing epigenetic plasticity. On the basis of this recognition, we hypothesized that small-molecule activators of toll-like receptor 3, together with external microenvironmental cues that drive endothelial cell (EC) specification, might be sufficient to induce transdifferentiation of fibroblasts into ECs (induced ECs). Methods and Results— We show that toll-like receptor 3 agonist Poly I:C, combined with exogenous EC growth factors, transdifferentiated human fibroblasts into ECs. These induced ECs were comparable to human dermal microvascular ECs in immunohistochemical, genetic, and functional assays, including the ability to form capillary-like structures and to incorporate acetylated low-density lipoprotein. Furthermore, induced ECs significantly improved limb perfusion and neovascularization in the murine ischemic hindlimb. Finally, using genetic knockdown studies, we found that the effective transdifferentiation of human fibroblasts to ECs requires innate immune activation. Conclusions— This study suggests that manipulation of innate immune signaling may be generally used to modify cell fate. Because similar signaling pathways are activated by damage-associated molecular patterns, epigenetic plasticity induced by innate immunity may play a fundamental role in transdifferentiation during wound healing and regeneration. Finally, this study is a first step toward development of a small-molecule strategy for therapeutic transdifferentiation for vascular disease.

Rationale: We have previously shown that innate immunity is necessary for transdifferentiation of fibroblasts to endothelial cells. A major signaling molecule involved in innate immunity is inducible nitric oxide synthase (iNOS). Accordingly, we hypothesized that iNOS-generated nitric oxide (NO) might enhance transdifferentiation. Objective: To elucidate the role of NO in epigenetic plasticity during transdifferentiation. Methods and Results: We exposed the BJ fibroblasts to transdifferentiation formulation that included endothelial growth factors and innate immune activator polyinosinic:polycytidylic acid to induce endothelial cells. Generation of transdifferentiated endothelial cells was associated with iNOS expression and NO elaboration. In the absence of polyinosinic:polycytidylic acid, or in the presence of antagonists of NFκB (nuclear factor kappa B) or iNOS activity, NO synthesis and induce endothelial cell generation was reduced. Furthermore, genetic knockout (in murine embryonic fibroblasts) or siRNA knockdown (in BJ fibroblasts) of iNOS nearly abolished transdifferentiation, an effect that could be reversed by iNOS overexpression. Notably, polyinosinic:polycytidylic acid induced nuclear localization of iNOS, and its binding to, and nitrosylation of, the epigenetic modifier ring finger protein 1A (RING1A) as assessed by immunostaining, Co-IP, and mass spectrometry. Nitrosylation of RING1A reduced its binding to chromatin and reduced global levels of repressive histone marker H3K27 trimethylation. Overexpression of a mutant form of RING1A (C398A) lacking the nitrosylation site almost abrogated transdifferentiation. Conclusions: Overall, our data indicate that during transdifferentiation, innate immune activation increases iNOS generation of NO to S-nitrosylate RING1A, a key member of the polycomb repressive complex. Nitrosylation of RING1A reduces its binding to chromatin and decreases H3K27 trimethylation level. The release of epigenetic repression by nitrosylation of RING1A is critical for effective transdifferentiation.

We thank Drs Li and Hao for their thoughtful comments on our recent article on the role of innate immunity in transdifferentiation of human fibroblasts to endothelial cells.1 They correctly state that the epigenetic landscape is different from one somatic cell to another and that the determinants of that epigenetic landscape (such as small RNAs, transcriptional factors, and chromatin modifiers) also differ from one somatic cell to another. Accordingly, they pose an excellent question: Because of the known differences between somatic cells in their epigenetic landscape and …