Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

Reviewer #1 (Public review):

Summary:

This manuscript analyses primarily the effects of deleting the TgfbR1 and TgfbR2 receptors from endothelial cells at postnatal stages of vascular development and blood-retina barrier maturation in the retina. The authors find that deletion of these receptors affects vascular development in the retina, but importantly, it affects the infiltration of immune cells across the vessels in the retina. The findings demonstrate that Tgfb signaling through TgfbR1/R2 heterodimers regulates primarily the immune phenotypes of endothelial cells in addition to regulating vascular development. The data provided by the authors provide a solid support for their conclusions.

Strengths:

(1) The manuscript uses a variety of elegant genetic studies in mice to analyze the role of TgfbR1 and TgfbR2 receptors in endothelial cells at postnatal stages of vascular development and blood-retina barrier maturation in the retina.

(2) The authors provide a nice comparison of the vascular phenotypes in endothelial-specific knockout of TgfbR1 and TgfbR2 in the retina (and to a lesser degree in the brain) with those from Npd KO mice (loss of Ndp/Fzd signaling) or loss of VEGF-A signaling to dissect the specific roles of Tgf signaling for vascular development in the retina.

(3) The snRNAseq data of vessel segments from the brains of WT versus TgfbR1 -iECKO mice provides a nice analysis of pathways and transcripts that are regulated by Tgfb signaling in endothelial cells.

Weaknesses:

(1) The authors claim that choroidal neovascular tuft phenotypes are similar in TgfbrR1 KO and TgfbrR2 KO mice. However, the phenotypes look more severe in the TgfbrR1 KO rather than TgfbrR2 KO mice. Can the authors show a quantitative comparison of the number of choroidal neovascular tufts per whole eye cross-section in both genotypes?

(2) In the analysis of Sulfo-NHS-Biotin leakage in the retina to assess blood-retina barrier maturation. The authors claim that there is increased vascular leakage in the TgfbR1 KO mice. However, it does not seem like Sulfo-NHS-biotin is leaking outside the vessels. Therefore, it cannot be increased vascular permeability. Can the authors provide a detailed quantification of the leakage phenotype?

(3) The immune cell phenotyping by snRNAseq is premature, as the number of cells is very small. The authors should sort for CD45+ cells and perform single-cell RNA sequencing.

(4) The analysis of BBB leakage phenotype in TgfbR1 KO mice needs to be more detailed and include tracers as well as serum IgG leakage.

(5) A previous study (Zarkada et al., 2021, Developmental Cell) showed that EC-deletion of Alk5 affects the D tip cells. The phenotypes of those mice look very similar to those shown for TgfbrR1 KO mice. Are D-tip cells lost in these mutants by snRNAseq?

Reviewer #2 (Public review):

Summary:

The authors meticulously characterized EC-specific Tgfbr1, Tgfbr2, or double knockout in the retina, demonstrating through convincing immunostaining data that loss of TGF-β signaling disrupts retinal angiogenesis and choroidal neovascularization. Compared to other genetic models (Fzd4 KO, Ndp KO, VEGF KO), the Tgfbr1/2 KO retina exhibits the most severe immune cell infiltration. The authors proposed that TGF-β signaling loss triggers vascular inflammation, attracting immune cells - a phenotype specific to CNS vasculature, as non-CNS organs remain unaffected.

Strengths:

The immunostaining results presented are clear and robust. The authors performed well-controlled analyses against relevant mouse models. snRNA-seq corroborates immune cell leakage in the retina and vascular inflammation in the brain.

Weaknesses:

The causal link between TGF-β loss, vascular inflammation, and immune infiltration remains unresolved. The authors' model posits that EC-specific TGF-β loss directly causes inflammation, which recruits immune cells. However, an alternative explanation is plausible: Tgfbr1/2 KO-induced developmental defects (e.g., leaky vessels) permit immune extravasation, subsequently triggering inflammation. The observations that vein-specific upregulation of ICAM1 staining and the lack of immune infiltration phenotypes in the non-CNS tissues support the alternative model. Late-stage induction of Tgfbr1/2 KO (avoiding developmental confounders) could clarify TGF-β's role in retinal angiogenesis versus anti-inflammation.