Novel Apelin-expressing gCap Endothelial Stem-like Cells Orchestrate Lung Microvascular Repair

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    eLife Assessment:

    The manuscript by Godoy and colleagues is an important contribution to the understanding of how lung endothelial regeneration progresses following endothelial ablation. The novelty and elegance of this study are rooted in the regional and specific ablation of lung endothelial cells using diphtheria toxin without the massive inflammatory activation that is seen with lung injury induced by bacterial infections, viral infections, or lipopolysaccharide. The data convincingly demonstrate that there is an emergence of a highly proliferative lung endothelial subpopulation that drives endothelial regeneration. The translational implications of the study include the identification of potential therapeutic targets to augment endothelial regeneration as a treatment for ALI/ARDS. This study will be of interest to vascular biologists, lung biologists, and researchers studying adult tissue regeneration.

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Abstract

Question

We sought to define the mechanism underlying lung microvascular regeneration in a severe acute lung injury (ALI) model induced by selective lung endothelial cell ablation.

Methods

Changes in lung cell populations and gene expression profiles were determined in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs using single-cell RNA sequencing at baseline (day 0) and days 3, 5 and 7 after lung EC ablation.

Results

Eight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline, and general capillary (gCap) ECs expressing the apelin receptor. Intratracheal instillation of DT resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. At 3 days post injury, a novel gCap population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned to proliferative ECs, expressing apelin receptor together with the pro-proliferative transcription factor, FoxM1 . This progenitor-like cell population was responsible for the rapid replenishment of all depleted EC populations by 7 days post injury, including aerocytes which play a critical role in re-establishment of the air-blood barrier. Treatment with an apelin receptor antagonist prevented recovery and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair.

Conclusion

The lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by signaling between newly emergent apelin-expressing gCap endothelial stem-like cells and highly proliferative, apelin receptor positive endothelial progenitors.

Take-Home message

Using sublethal lung endothelial cell (EC) ablation, we show for the first that EC regeneration and resolution of acute lung injury is orchestrated by novel apelin-expressing, gCap endothelial stem-like cells by a mechanism requiring apelin signaling.

Graphical Abstract

A schematic representation of EC populations contributing to microvascular repair. At baseline (Day 0), there are two main alveolar groups of capillary ECs: larger apelin positive aCap ECs, termed aerocytes, that play a key structural role in forming the air-blood barrier; and smaller apelin receptor ( Aplnr ) expressing gCap ECs, which are found in the thicker regions at the corners of the alveoli. After DT-induced EC ablation, there is a marked depletion of both EC populations and the appearance of novel transitional and transient populations. At Day 3, there is the appearance of stem-like gCap ECs that paradoxically express apelin, but not its receptor, and are characterized by various stem and progenitor cell markers but show no evidence of proliferation. By Day 5, these transition to ECs expressing Aplnr which have a strong proliferative phenotype, as evidenced by FoxM1 and Ki67 expression, and then rapidly replenish depleted EC pools, including aCap ECs, by Day 7. This transition is orchestrated by the interaction of apelin with its receptor as a critical mechanism in lung microvascular regeneration after EC injury. AT1 = alveolar type −1 epithelial cell; AT2 = alveolar type-2 epithelial cell; APLNR = apelin receptor; ANGPT2 = angiopoietin 2; EPCR = Endothelial protein C receptor.

Article activity feed

  1. eLife Assessment:

    The manuscript by Godoy and colleagues is an important contribution to the understanding of how lung endothelial regeneration progresses following endothelial ablation. The novelty and elegance of this study are rooted in the regional and specific ablation of lung endothelial cells using diphtheria toxin without the massive inflammatory activation that is seen with lung injury induced by bacterial infections, viral infections, or lipopolysaccharide. The data convincingly demonstrate that there is an emergence of a highly proliferative lung endothelial subpopulation that drives endothelial regeneration. The translational implications of the study include the identification of potential therapeutic targets to augment endothelial regeneration as a treatment for ALI/ARDS. This study will be of interest to vascular biologists, lung biologists, and researchers studying adult tissue regeneration.

  2. Reviewer #1 (Public Review):

    This study sought to establish a model of targeted lung endothelial ablation and subsequently study the regeneration process post-ablation using single-cell RNA-sequencing in order to identify key subpopulations and underlying mechanisms of regeneration.

    Strengths of the study include:

    1. The elegance of the DT endothelial ablation model which leverages local lung instillation of DT to locally ablate the endothelium and cause significant lung vascular leakiness while keeping the endothelium of other organs intact, as is convincingly demonstrated in Fig 1 and Fig 2.

    2. The temporal analyses using scRNA-seq demonstrate key shifts in endothelial and non-endothelial cell populations following endothelial injury. These experiments identify a highly proliferative subpopulation of endothelial cells that expresses the transcription factor FoxM1 during the regeneration phase.

    3. The authors discover that the traditionally designated "gCap" lung endothelial population contains additional subpopulations that have regenerative potential and that there is a transient expression of apelin in the regenerative population. Pharmacological inhibition of the apelin receptor increase mortality.

    Potential weaknesses include:

    1. The description of the "stem-like" nature of endothelial cells is not experimentally proven. "Stem-like" is a vague term and the usage of this term is primarily based on the expression of Procr. However, that itself does not justify the usage of "stem-like" unless there is more clear evidence of what "stem-like" properties these cells have, such as multipotency.

    2. The intriguing finding of the proliferative EC population raises the question as to how these cells emerge. Do they have a specific subpopulation/cluster origin in the baseline lung endothelium, and was Apelin expression both necessary as well as sufficient to induce the switch to the proliferative state? Such mechanistic analyses would be very helpful in understanding the coordination of the lung endothelial regeneration program.

    3. The authors mention that endothelial ablation also induces shifts in the numbers of other cell types such as epithelial cells, alveolar macrophages, and immune cells but there is no analysis beyond the quantification of the cells. Are these cells involved in the regeneration of the endothelium by providing ligands such as growth factors?

  3. Reviewer #2 (Public Review):

    Acute lung injury (ALI) and ARDS are major causes of morbidity and mortality in critically ill patients and patients infected with Sars-Cov-2. There are no effective therapies for ALI/ARDS, and the 28-day mortality rate is ~40%. One of the main pathological features of ALI/ARDS is a vascular injury characterized by endothelial dysfunction, inflammation, and in situ thrombosis. Using a murine model of ALI/ARDS triggered by diphtheria toxin (DT) mediated endothelial specific ablation, the authors apply sc-RNA-seq analysis to study how lung cell populations respond to injury and identify two main endothelial subpopulations responsible for regenerating lung vasculature over seven days. The study's implications are exciting as they provide evidence of intrinsic repair mechanisms that could be targeted for vascular regeneration and recovery of lung function in the context of ALI/ARDS. In particular, the apelin pathway rises as a prime therapeutic candidate given its role in coordinating the behavior of general and aerocyte capillary cells in lung vascular repair.

    While the results of this study are exciting and novel, it must be recognized that several limitations need to be properly addressed to facilitate the translation of the findings toward medical care. For instance, the animal model used in this study (DT mediated EC ablation) does not fully recapitulate all the pathological hallmarks of ALI/ARDS, the most important of which is that repair proceeds at a very slow pace as a result of multiple factors that are not recapitulated in this made. Since the authors use only one model of ALI/ARDS, it is not entirely clear whether the current findings can be generalized to other models. Since no one model truly recapitulates the complexity of human ALI/ARDS, it is important to use at least two or more models that can narrow genetic and molecular mechanisms fundamental to lung injury and recovery. Another important aspect is the lack of validation in human samples and cells, which could strengthen the conclusions raised by the authors in the discussion. Finally, the authors appropriately emphasize how this study could help efforts to understand Sars-Cov2 mediated ALI/ARDS. Still, no studies explore any overlap with currently available Omics data from COVID lungs.

    Despite these weaknesses, this study is the first to apply rigorous scRNA-seq analysis to this unique model of ALI/ARDS. It also provides data to support the importance of the two newly discovered endothelial cell subpopulations (gCap and aCap) in lung repair and regeneration, which hold the potential to offer unique mechanistic insights into the genetic and molecular mechanisms responsible for vascular repair and offers the opportunity to consider apelin based therapeutic approaches to treat ALI/ARDS. In conclusion, this study is expected to contribute to our lung biology understanding greatly. It provides the research community with novel resources and tools that greatly aid efforts to understand ALI/ARDS and identify therapeutics to treat this devastating disease.

  4. Reviewer #3 (Public Review):

    This highly innovative study makes elegant use of single-cell RNA sequencing in a transgenic murine model of selective lung endothelial depletion to study endothelial repair and regeneration. Within 3 days after ablation of 70% of lung endothelial cells, a new stem-like endothelial population expressing markers of general capillary endothelial cells (gCap), yet also apelin, Procr, Angpt2, and CD93, yet not the gCap-typical apelin receptor emerged. This was followed at day 5 by a population of highly proliferative gCap-like endothelial cells expressing the apelin receptor along with FoxM1, which replenished all depleted endothelial populations and allowed for rapid resolution of microvascular injury. These newly identified cell states are highly reminiscent of tip and stalk cells in sprouting angiogenesis and may guide the development of new regenerative strategies.

    Strengths:
    The present work provides important novel insights into the mechanisms of endothelial repair and reconstitution. Importantly, the authors identify a subset of gCap cells that upon endothelial depletion develops into a stem cell-like population expressing (among others) apelin, which signals via the apelin receptor to another, progenitor-like cell population that arises subsequently from the former stem cell-like population. These findings shed new light on the process of microvascular "healing" in acute lung injury and ARDS, and open up intriguing parallels to processes well known from angiogenic sprouting that may be exploited for therapeutic purposes.

    Weaknesses:
    As with every innovative study, the emerging answers give rise to a series of new questions. Notable among those is the identity of the signal that initially drives the transition of the stem cell-like gCap population from their basal state - the recognition of such a signal may allow replicating the proposed cycle in vitro, with the opportunity to harvest cells at specific time points for both research and therapeutic purposes. Similarly, one may wonder how a lung may survive with 70% of its endothelial cells gone - do the respective vascular segments simply get excluded from perfusion (and, possibly, ventilation, as AT-II cells also decline in parallel, resulting in an emphysematous phenotype) or does fluid simply leak into the interstitium (which seems hard to reconcile with survival)? From a methodological point of view, RNA velocity analyses may be considered in follow-up studies to further substantiate the notion of a gradual transition of a subset of gCap cells from a basal to a stem cell-like to a progenitor-like and back to a basal state.