Safeguarding the Vasculature: Redundant transcriptional machinery ensures endothelial protection

An intact and properly functioning hemovascular system is essential for survival in advanced vertebrates. Through extensive crosstalk between circulating (e.g. innate immune) and luminal (e.g. endothelial) cells, the hemovascular system ensures efficient and uninterrupted nutrient distribution, oxygenation, and widespread immunological protection. Disruption of the delicate balance that exists within this system leads to potentially disastrous consequences. Indeed, improper activation of any cellular component of the hemovascular system directly contributes to atherosclerotic, thrombotic, and ischemic disease. As such, mechanisms that promote quiescence and safeguard against inflammatory activation have evolved to preserve vascular integrity. This is conferred through the precise coordination of transcription factor networks, a system that contains substantial redundancy to increase the threshold for activation.

In this issue of ATVB, Lu and colleagues explore transcriptional redundancy in endothelial cells (ECs) by dissecting MEF2-mediated signaling pathways that maintain vascular health¹. Utilizing mice with combined endothelial deletion of Mef2a, Mef2c, and Mef2d (ACDiEKO), the authors demonstrated an absolute requirement for the MEF2 transcriptional network for maintenance of an intact vasculature. Remarkably, mice lacking two out of the three MEF2 family members were viable and did not demonstrate the profound vascular phenotype seen in the triple knockout, further illustrating the compensatory nature of a subset of endothelial transcription factors. Mechanistically, the authors identified multiple putative axes through which MEF2 achieves its protective functions. Among these is through direct transcriptional control of Krüppel-like factors (KLFs) 2 and 4. Indeed, ACDiEKO mice expressed KLF2/4 at essentially knock out levels, consequently affecting the transcription of hundreds of genes and numerous endothelial functions. Independent of KLF2/4, Lu et al. demonstrate that MEF2 also regulates Notch1 and YAP/TAZ signaling to affect vascular homeostasis. Altogether, this work expands on the field’s understanding of the complex transcriptional networks that exist in maintaining proper vascular function.

This work also underscores the importance of redundancy in ensuring vital physiologic processes such as proper delivery of nutrients and defense. Phenotypically, the ACDiEKO mouse is astonishingly similar to combined knockout of endothelial KLF2 and KLF4 (KLF2/4iEKO). Recent work from our group described a functional redundancy in which loss of either KLF2 or 4 was insufficient to compromise an intact vasculature². In fact, the presence of a single allele of either factor was sufficient to rescue the KLF2/4iEKO phenotype. Given that KLF2 and KLF4 expression are both decreased in the context of ACDiEKO, it is perhaps unsurprising that there is also substantial overlap in transcriptional effects between these two compound mutants. Further, the MEF2-KLF axis, itself, demonstrates redundancy as the presence of MEF2d, alone, is sufficient to maintain transcription of KLF2/4. To fully understand the KLF-dependent versus independent effects of MEF2 deletion, future studies will need to exogenously induce KLF2 or 4 expression in the context of MEF2 knockout. While the lethality of the ACDiEKO mouse may be rescued with restored KLF2/4 expression, there will likely still be numerous transcriptional effects due to the KLF-independent axes presented in this work (1).

This work and others that explore transcriptional redundancy illuminate a fundamental limitation in dissecting complex signaling networks via genetic knockout means. Single (or even double) knockout models do not necessarily illustrate the functions or importance of individual factors if their loss induces a compensatory response. It is important to note that while transcriptional redundancy by MEF2 or KLFs is needed for the most fundamental aspects of maintaining an intact vasculature for survival, lost expression of any single factor is not innocuous and can be pathological in response to provocative stimuli. Loss of endothelial KLF4, as an example, greatly increases susceptibility to atherothrombotic disease when challenged with metabolic stress or age, while loss of MEF2c increases susceptibility to intimal smooth muscle migration^3–5. The extent to which redundant transcriptional mechanisms protect against disease, therefore, is context dependent and needs further investigation.

In the present study, the authors combined sequencing datasets from multiple genetic and surgical perturbations in order to robustly explore the transcriptional overlaps that occur between loss-of-function models and disease. Through this, they have significantly added to the field’s understanding of redundancy in the pathogenesis of vascular inflammation. There still exists a gap in knowledge, however, on how these factors interact and affect each other’s functions during homeostasis. MEF2 and KLF2/4 are induced by protective stimuli such as laminar flow^{2, 6–9}. To understand how MEF2-KLF redundancy functions in homeostasis (versus as a failsafe against deleterious stimuli in disease), novel models will need to be devised that integrate tunable expression of combinations of factors with readouts such as occupancy dynamics between redundant family members and transcriptional effects of increased expression of components. This approach, paired with data from induced-expression studies (e.g. laminar flow), will give a complete picture of the relative importance of each factor in specific physiological states.

Finally, there is an exciting potential that these transcriptional redundancies may exist in close cellular relatives of ECs as well. Like ECs, myeloid cells (e.g. macrophages) rely on KLF2/4 transcription to maintain quiescence and prevent aberrant inflammatory activation^10–13. Further, MEF2 plays an important role in mediating transcription of macrophage KLF2¹⁴. While targeted experiments exploring KLF2/4 redundancy have not been performed, data from numerous studies would suggest a transcriptional and functional overlap in macrophages^{10, 12, 13, 15, 16}. Future insights into co-evolved transcriptional redundancy of components of the hemovascular system will undoubtedly advance overall understanding of how these cells interact and how they may be targeted for therapeutic gain.

Mechanisms of transcriptional redundancy in endothelial homeostasis.

Laminar shear stress (LSS) induces numerous protective gene programs, in part through the induction of Myocyte Enhancer Factor (MEF) signaling. Lu et al. demonstrated redundancy in the ability for MEF2a, c, or d in mediating MEF2’s transcriptional effects. Among these effects is the induction of Krüppel-like factor (KLF) signaling. Previous reports demonstrated redundancy between KLF2 and 4 in maintaining vascular integrity and Lu et al. advance insights on the MEF2-KLF axis by demonstrating shared transcriptional signatures. MEF2 also has KLF-independent transcriptional regulation as well through the regulation of Notch1 and YAP/TAZ signaling.