Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling
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Abstract
Pulmonary arterial hypertension (PAH) is a fatal disease characterized by pathological pulmonary artery remodeling. Endothelial cells (EC) injury including DNA damage is critically involved in the vascular remodeling in PAH, and persistent injury leads to cellular senescence in ECs. Here, we show that EC senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling. EC-specific progeroid mice that we recently generated showed exacerbated pulmonary hypertension after chronic hypoxia exposure, accompanied by the enhanced pulmonary arterial smooth muscle cells (PASMCs) proliferation in the distal pulmonary arteries. Mechanistically, we identified that senescent ECs highly expressed Notch ligands, and thus activated Notch signaling in PASMCs, leading to enhanced PASMCs proliferation and migration capacities. Consistently, pharmacological inhibition of Notch signaling attenuated the effects of senescent ECs on SMCs functions in vitro , and on the pulmonary hypertension in EC-specific progeroid mice in vivo . These data establish EC senescence as a crucial disease-modifying facor in PAH.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 29 2021, follows.
Summary
This paper builds on recent studies that have made the connection between chronic endothelial damage and cellular senescence among endothelial cells in PH. Here, using a transgenic mouse that expresses in endothelial cells a dominant negative form of the TRF2 protein needed for telomere maintenance, the authors induce cellular senescence in the endothelium and show that these mice demonstrate worse PH characteristics following exposure to chronic hypoxia. They go on to test the effect of this dominant negative protein on human pulmonary artery endothelial cells in vitro and show that transfected ECs increase expression of secreted and surface-bound signaling molecules, and that when co-cultured in …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 29 2021, follows.
Summary
This paper builds on recent studies that have made the connection between chronic endothelial damage and cellular senescence among endothelial cells in PH. Here, using a transgenic mouse that expresses in endothelial cells a dominant negative form of the TRF2 protein needed for telomere maintenance, the authors induce cellular senescence in the endothelium and show that these mice demonstrate worse PH characteristics following exposure to chronic hypoxia. They go on to test the effect of this dominant negative protein on human pulmonary artery endothelial cells in vitro and show that transfected ECs increase expression of secreted and surface-bound signaling molecules, and that when co-cultured in direct contact with pulmonary artery smooth muscle cells the SMCs increase their proliferation, an effect blocked by pharmacologic inhibition of Notch signaling. Notch blockade in vivo attenuates pulmonary hypertension in both transgenics and wild type controls. These data provide an intriguing framework for understanding how endothelial damage alters signaling to neighboring cells in the vascular wall and provides further evidence that Notch signaling plays a key role in the development of PH vasculopathies.
Essential Revisions
It's unclear where within the pulmonary vasculature the TRF2DN transgene is expressed, and therefore which vessels are effected by senescence. That the transgene is driven by a well-established endothelial promoter (VEcad) is not sufficient to demonstrate universal expression. Especially in the case of a transgene whose expression is intended to result in chromosomal abnormalities, DNA damage and a halt to cell proliferation, significant mosaicism is to be expected. In situ hybridization with probes specific to TRF2DN or an antibody stain that specifically recognizes the transgenic protein on lung tissue sections would address this problem. Both representative images and a careful characterization of the classes of arteries (subdivided by diameter, for example), capillaries, veins, and lymphatics that express the transgene and with which levels of mosaicism would be ideal.
Validation of the EC expression changes, specifically of the Notch ligands identified as upregulated in vitro, need to be validated in situ to ensure they are upregulated in the endothelium of arteries where the PH phenotype (increased muscularization, increased SMC proliferation) is observed in this model. Whole lung dissociation followed by enrichment for CD146+ ECs will result in an overwhelming number of capillary ECs and a tiny number of artery ECs (Figure 3E). Similarly for the in vivo validation of Notch reception in SMCs through qPCR for indicators of Notch reception (Figure 3F, 4I) - this experiment was done on whole lung lysate and does not demonstrate increased expression of these genes in the artery wall. In situ hybridization with probes specific to TRF2DN or an antibody stain that specifically recognizes the transgenic protein on lung tissue sections would address this problem. Both representative images and a careful characterization of the classes of arteries (subdivided by diameter, for example), capillaries, veins, and lymphatics that express the transgene and with which levels of mosaicism would be ideal.
The method by which PAs are identified (Figure 1D, 4F) and the metrics by which artery muscularization from images of tissue sections is quantitated (Figure 1F, 1H, 4H) are somewhat unclear and appear to be made from very few fields from an unspecified number of animals. There appears to be significant variance in artery response to hypoxia (comparing Figure 1E with vehicle in 4G), which is not a problem and very much to be expected, but means there must be absolute clarity in how the data for graphs summarizing imaging data were obtained. A supplementary figure with representative images demonstrating how arteries were scored would be very helpful. The number of independent mice for each analysis must appear either in the figure legends or in the relevant sections of the methods. A reader's understanding of how the PAs were identified in Figure 1D and 4F would be helped by using a vascular specific antibody stain. And a supplementary figure with a large panel of artery images from Tg and Wt animals before and after hypoxia exposure, with and without DAPT, so the reader can grasp the range of effects on vessels in each case would be immensely helpful.
Please describe the in vivo relevance of endothelial progeria induced by decreased TERF2 function in patients with PAH.
While endothelial senescence leads to decreased proliferation and apoptosis of EC, which have been shown to occur in PAH, clonal proliferation of EC is also a hallmark of advanced disease in PAH. The study does not comment on this varied phenotype of EC in the pulmonary circulation in PAH patients and the relationship of senescence of EC to SMC migration.
Increased levels of Jag1 have been linked to excess proliferation in several cancer cell lines. In the context of senescence with decreased EC proliferation, increase in Jag1/Jag2 levels is surprising and the paper does not comment on this phenotype as being distinct from cancer cells.
The mechanism for increased notch ligand expression in response to progeria was indirectly addressed with 5-Aza studies which presumably leads to inhibition of DNA methylation. However, it is unclear how this inhibits increase in notch ligand expression. In the discussion, the authors mention (Line 17, page 10) that DNA hypomethylation promotes specific transcriptional programs as a result of senescence. However, 5-Aza prevented the induction of Notch ligand expression in senescent EC (Suppl Fig 2). The discussion of these results needs further clarification. It is unclear what specific epigenetic modifications occur to increase the expression of Jag1/2 and Dll4 in senescence associated changes.
The study did not report whether aorta and other systemic vessels demonstrate senescence changes in endothelial cells-endothelial progeria in the TG mice would involve all vasculature. Presumably, vascular remodeling was limited to the lung, given the unique response of the lung to hypoxia. However, examination of a systemic vascular bed would have strengthened the conclusions of the study. Do the EC and SMC derived from aorta or coronary vessels show similar responses in vitro compared to human PAEC with DN-TERF2 transfection?
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