# Venous Endothelial Cells Promote Osteoblast Differentiation More Effectively Than Arterial Cells via TGF‐β/BMP9 and Notch Pathway‐Related Gene Expression

## TL;DR

Venous endothelial cells better support bone cell development than arterial cells by activating specific signaling pathways and improving the bone environment.

## Contribution

The study reveals that venous endothelial cells promote osteoblast differentiation more effectively than arterial cells through distinct paracrine signaling.

## Key findings

- Venous endothelial cell conditioned media significantly enhance osteoblast differentiation and mineralization.
- HUVECs activate TGF-β/BMP9 and Notch pathways, promoting cytoskeletal remodeling and ECM organization.
- Venous endothelial cells induce a hypoxia–VEGF feedback, supporting angiogenic–osteogenic coupling.

## Abstract

The coupling between angiogenesis and osteogenesis is a key determinant of skeletal homeostasis, yet the influence of endothelial cell origin on osteoblast differentiation remains underexplored. Here, we investigated how venous (HUVECs) and arterial (HCAECs) endothelial cells differentially modulate the osteogenic phenotype of human osteoblasts via paracrine signaling. To better address this issue, the conditioned media (CM) from HUVECs significantly enhanced osteoblast differentiation, as evidenced by increased alkaline phosphatase activity, upregulation of canonical markers such as Runx2, Osterix, and Osteocalcin, and activation of matrix mineralization genes (Tnap, Bsp, Col1a1). In contrast, CM from HCAECs induced a markedly weaker response. qPCR analysis revealed that HUVEC‐CM robustly stimulated key osteoinductive pathways, including TGF‐β/BMP9 and Notch, with pronounced activation of SMADs, Jagged, and Notch receptors. Moreover, HUVEC‐CM promoted cytoskeletal remodeling via increased expression of Integrin‐β1, FAK, Src, and Cofilin, and favored ECM organization by repressing MMP activity and enhancing Reck expression. Hypoxia‐associated markers (Hif1α, Vegf) were also elevated in HUVEC‐treated osteoblasts, supporting enhanced angiogenic‐osteogenic coupling. Principal component and network analyses confirmed a distinct molecular clustering for HUVEC‐responsive genes. Altogether, our data demonstrate that venous endothelial cells, through their specific secretome, provide a more adequate microenvironment for osteoblast differentiation and mineralization compared to their arterial equivalent. These findings underscore the functional relevance of endothelial plasticity in bone regeneration and support the use of venous‐derived endothelial factors in bone tissue engineering strategies.

Bone regeneration depends on precise coupling between angiogenesis and osteogenesis, yet how endothelial origin shapes osteoblast fate is unclear.Here we show that venous endothelial cells (HUVECs) secrete factors that outperform arterial cells (HCAECs) in driving osteoblast differentiation and matrix mineralization, via coordinated activation of TGF‐β/BMP9–SMAD and Notch axes, reinforcement of focal‐adhesion/cytoskeletal programs, and induction of a hypoxia–VEGF feedback.These data identify a venous‐biased paracrine signature that aligns with bone microvascular biology and offers a clear criterion for selecting endothelial sources or secretomes to enhance bone tissue engineering and repair.

Bone regeneration depends on precise coupling between angiogenesis and osteogenesis, yet how endothelial origin shapes osteoblast fate is unclear.

Here we show that venous endothelial cells (HUVECs) secrete factors that outperform arterial cells (HCAECs) in driving osteoblast differentiation and matrix mineralization, via coordinated activation of TGF‐β/BMP9–SMAD and Notch axes, reinforcement of focal‐adhesion/cytoskeletal programs, and induction of a hypoxia–VEGF feedback.

These data identify a venous‐biased paracrine signature that aligns with bone microvascular biology and offers a clear criterion for selecting endothelial sources or secretomes to enhance bone tissue engineering and repair.