CD146⁺ endothelial and pericyte populations define a repair-associated neovascularization program in rheumatoid arthritis pannus

Technical Summary: CD146⁺ Endothelial and Pericyte Populations Define a Repair-Associated Neovascularization Program in Rheumatoid Arthritis Pannus

Problem Statement
Rheumatoid arthritis (RA) is characterized by the formation of pannus, a hyperplastic synovial tissue driven by inflammation and extensive neovascularization. While neovascularization is known to facilitate inflammatory cell infiltration and nutrient supply, the specific cellular programs and heterogeneity of the vascular cells (endothelial cells and pericytes) underlying this pathological angiogenesis remain incompletely understood. Specifically, the distinct roles of pericyte subsets and their interaction with endothelial cells in the context of chronic synovial inflammation require further elucidation.

Methodology
The study utilized the D1BC transgenic mouse model, which develops chronic, severe polyarthritis resembling human RA following low-dose bovine type II collagen administration. The research employed a multi-modal approach:

1. Flow Cytometry and Bulk RNA-seq: Synovial cells from inflamed joints were isolated and cultured briefly. Cells were sorted into four populations based on CD146 (MCAM) and CD140a (PDGFRα) expression: CD146^high^, CD146^mid^, CD140a^+^, and double-negative. Bulk RNA sequencing was performed to characterize transcriptomic profiles.
2. Single-Cell RNA Sequencing (scRNA-seq): CD146^+^ synovial cells were enriched using magnetic beads and subjected to scRNA-seq using the TAS-seq protocol on the BD Rhapsody system. Data were analyzed using Seurat, Slingshot (for trajectory analysis), and LIANA (for ligand-receptor interaction prediction).
3. Comparative Analysis: The mouse datasets were integrated with publicly available human scRNA-seq datasets from RA synovium and lung cancer to identify conserved cell states.
4. Validation: Immunohistochemistry (IHC) was performed on joint sections to visualize the spatial distribution of CD31 (endothelium), NG2/Cspg4, PDGFRβ, and CD146. RT-qPCR validated gene expression levels in tissue samples.

Key Results

• Identification of Vascular Subsets: Bulk RNA-seq revealed that CD146^high^ cells are enriched for endothelial markers (e.g., Icam2, Cd151, Apln, Ednrb), while CD146^mid^ and CD140a^+^ populations express pericyte markers (Pdgfrb, Acta2) and osteochondrogenic markers.
• Endothelial Heterogeneity: scRNA-seq identified four distinct endothelial subsets defined by Apln and Aplnr expression: Apln^+^, Aplnr^+^, Apln^+^/Aplnr^+^, and double-negative. The Apln^+^/Aplnr^+^ double-positive population exhibited transcriptional features consistent with repair-associated capillary programs, sharing similarities with endothelial states observed during pulmonary capillary regeneration. These cells expressed neovascularization genes (Angpt2, Cd34, Kdr, Cldn5, Procr) but lacked gas-exchange markers typical of mature aerocytes.
• Distinct Pericyte Populations: The study identified a unique CD146^+^ pericyte population (clusters c6, c11, c19) that expressed Cspg4 (NG2) and Fgf5 but lacked Pdgfrb and Acta2. This population, termed "type-2 pericytes," also expressed progenitor-associated genes (Stc2, Sox2, Sox10). In contrast, "type-1 pericytes" (cluster c3) expressed Pdgfrb and Acta2.
• Cell-Cell Communication: Ligand-receptor analysis (LIANA) indicated active communication between the CD146^+^ endothelial cells and the Pdgfrb-negative type-2 pericytes. Key signaling pathways included:
  • Notch Signaling: Endothelial cells expressed Dll4 and Jag1, interacting with Notch4 on type-2 pericytes.
  • Slit-Robo Axis: Type-2 pericytes expressed Slit3, interacting with Robo4 on endothelial cells.
  • Apln-Aplnr: Autocrine/paracrine signaling within endothelial subsets.
• Spatial Validation: Immunohistochemistry confirmed that NG2^+^ pericytes are spatially associated with CD31^+^ neovessels within the pannus, though co-localization with PDGFRβ was limited, supporting the existence of the distinct NG2^+^/PDGFRβ^−^ population.
• Conservation: Similar CD146^+^ endothelial and pericyte subpopulations were identified in human RA and lung cancer datasets, suggesting a conserved repair-associated vascular program.

Significance and Claims
The authors claim that their findings identify distinct CD146^+^ endothelial and pericyte populations that characterize a "repair-associated neovascularization program" within the rheumatoid arthritis pannus. The study posits that pannus neovascularization shares molecular features with vascular regeneration programs activated during tissue injury, specifically resembling pulmonary capillary repair.

The paper highlights that the CD146^+^ endothelial cells and the distinct NG2^+^/PDGFRβ^−^ type-2 pericytes form a coordinated stromal-neovascular network. The authors suggest that these interactions, mediated by pathways such as Notch, Slit-Robo, and Apln-Aplnr, are potential candidates for selective modulation of pathological neovascularization in RA. The study concludes that these cellular populations provide new insight into the mechanisms of pathological angiogenesis in chronic synovial inflammation.

Limitations
The authors acknowledge that synovial cells were cultured briefly prior to sequencing due to limited cell yield from murine joints, which may introduce culture-induced changes, although bulk and single-cell comparisons suggested minimal effects. Furthermore, conclusions are primarily based on transcriptomic data with qualitative protein validation via IHC; functional significance requires further investigation.