The REFLEX system enables in vivo identification of perivascular angiogenic macrophages in the heart

Technical Summary: The REFLEX System for In Vivo Identification of Perivascular Angiogenic Macrophages

Problem Statement
A fundamental limitation in current biological research is the inability to directly identify physically interacting cells within living organisms (in vivo). Conventional interactome analyses rely on inferring signaling partners from bulk or single-cell transcriptomes; however, these methods cannot distinguish which cells are in direct physical contact versus those merely sharing a tissue microenvironment. This gap is particularly critical in the context of pressure-overload induced cardiac remodeling, where VEGF-A is known to be essential for maintaining vascular integrity and cardiac function. Despite this, the specific cellular source of VEGF-A and the precise mechanism by which the peptide is delivered to vascular endothelial cells remain undefined. Furthermore, conventional transcriptomic predictions have historically suggested cardiomyocytes as the dominant source of VEGF-A, a hypothesis that requires functional validation regarding its necessity in heart failure models.

Methodology
To address the challenge of mapping direct cell-to-cell contacts, the authors developed a genetically encoded platform integrating two distinct technologies:

1. REFLEX Mice: A genetically engineered mouse model designed to label cells that physically interact with a specific target cell type.
2. HUNTERuni-seq: A sequencing approach coupled with the REFLEX system to enable the unbiased detection and transcriptional profiling of the cells identified as interacting partners.

This combined approach allows for the isolation and characterization of cells that are in direct physical contact with vascular endothelial cells, bypassing the limitations of inferring interaction solely from gene expression data.

Key Contributions and Results
Using the REFLEX and HUNTERuni-seq platform, the study achieved the following:

• Identification of PVAMs: The system successfully identified a specific subpopulation of Vegfa-positive macrophages residing in close proximity to vascular endothelial cells. The authors named these cells perivascular angiogenic macrophages (PVAMs).
• Functional Necessity of PVAM-Derived VEGF-A: Despite the observation that the absolute amount of VEGF-A within PVAMs is small, the study demonstrated that the loss of VEGF-A specifically in PVAMs leads to impaired angiogenesis and reduced systolic function during pressure overload.
• Mechanism of Signal Delivery: The findings indicate that the direct physical contact between PVAMs and endothelial cells is a critical determinant for the effective delivery of VEGF-A. This suggests that VEGF-A signaling in this context does not rely on free diffusion through the interstitium.
• Refutation of Cardiomyocyte-Derived VEGF-A: Contrary to predictions made by conventional interactome analyses, which identified cardiomyocytes as the dominant source of VEGF-A, the authors found that deleting Vegfa specifically in cardiomyocytes had no measurable effect on capillary density or systolic function in a model of heart failure.

Significance
The paper establishes the REFLEX and HUNTERuni-seq platform as a versatile tool for uncovering biologically critical cell-to-cell interactions that are otherwise obscured by traditional transcriptomic inference. By shifting the focus from inferred signaling to physically verified contact, the study provides new insights into intercellular communication within pathological tissue contexts. Specifically, it redefines the understanding of VEGF-A signaling in cardiac remodeling, highlighting that cellular proximity and physical contact are more significant than free diffusion or the abundance of the ligand in the interstitium. These findings correct previous assumptions regarding the cellular sources of VEGF-A and underscore the importance of direct cell-cell contact in maintaining vascular integrity and cardiac function under stress.