The intricate cellular ecosystem of human peripheral veins as revealed by single-cell transcriptomic analysis

Abstract

The venous system has been historically understudied despite its critical roles in blood distribution, heart function, and systemic immunity. This study dissects the microanatomy of upper arm veins at the single cell level, and how it relates to wall structure, remodeling processes, and inflammatory responses to injury. We applied single-cell RNA sequencing to 4 non-diseased human veins (3 basilic, 1 cephalic) obtained from organ donors, followed by bioinformatic and histological analyses. Unsupervised clustering of 20,006 cells revealed a complex ecosystem of endothelial cell (EC) types, smooth muscle cell (SMCs) and pericytes, various types of fibroblasts, and immune cell populations. The venous endothelium showed significant upregulation of cell adhesion genes, with arteriovenous zonation EC phenotypes highlighting the heterogeneity of vasa vasorum (VV) microvessels. Venous SMCs had atypical contractile phenotypes and showed widespread localization in the intima and media. MYH11+DESlo SMCs were transcriptionally associated with negative regulation of contraction and pro-inflammatory gene expression. MYH11+DEShi SMCs showed significant upregulation of extracellular matrix genes and pro-migratory mediators. Venous fibroblasts ranging from secretory to myofibroblastic phenotypes were 4X more abundant than SMCs and widely distributed throughout the wall. Fibroblast-derived angiopoietin-like factors were identified as versatile signaling hubs to regulate angiogenesis and SMC proliferation. An abundant monocyte/macrophage population was detected and confirmed by histology, including pro-inflammatory and homeostatic phenotypes, with cell counts positively correlated with age. Ligand-receptor interactome networks identified the venous endothelium in the main lumen and the VV as a niche for monocyte recruitment and infiltration. This study underscores the transcriptional uniqueness of venous cells and their relevance for vascular inflammation and remodeling processes. Findings from this study may be relevant for molecular investigations of upper arm veins used for vascular access creation, where single-cell analyses of cell composition and phenotypes are currently lacking.

Fig 1. Wall structure and cell composition of upper arm veins.

A) Representative Masson’s trichrome stained cross-section of a basilic vein, indicating the intimal (I), medial (M), and adventitial (A) layers. Cells are stained in red while extracellular matrix appears in blue. Vasa vasorum are indicated by yellow arrowheads. B-C) Uniform manifold approximation and projection (UMAP) plot of 20,006 cells isolated from 4 veins (1 cephalic [CV1], 3 basilic [BV1-3]) and proportion of cell types per vein sample. D) Markers used for cell cluster identification.

Fig 2. Endothelial cells (EC) in upper arm veins.

A) Focused UMAP of 1,695 ECs isolated from upper arm veins. B) Feature plots indicating normalized expression levels of markers characteristic of the 5 EC populations. C-D) Identification of venous (PLVAP⁺ACKR1⁺) and arteriolar (PLVAP^-GJA5⁺) ECs in the main lumen of veins and vasa vasorum by immunofluorescence. Ven. = venule; Art. = arteriole. Scale bars represent 20 μm in all panels. E) Violin plot representation of expression differences among the 5 EC populations.

Fig 3. Smooth muscle cells (SMC) and pericytes in upper arm veins.

A) Focused UMAP of 2,298 SMCs isolated from upper arm veins. B) Feature plots indicating normalized expression levels of markers characteristic of the 3 SMC populations. C) Localization of SMC1 (MYH11⁺DES^hi) and SMC2 (MYH11⁺DES^lo) cells in the intima (I) and media (M) by immunofluorescence. Scale bars represent 20 μm in all panels. D) Violin plot representation of expression differences among the 3 SMC populations.

Fig 4. Fibroblasts in upper arm veins.

A) Focused UMAP of 9,172 fibroblasts isolated from upper arm veins. B) Localization of PDGFRA⁺ (pan-fibroblast marker) and APOD⁺ (secretory marker) fibroblasts by immunohistochemistry. Scale bars represent 50 μm. C) Feature plots indicating the relative expressions of fibroblast markers across a pseudotime defined by transcriptional similarities among cells. The Moran’s I statistics of spatial autocorrelation is shown for each gene. D) Gene ontology pathways unique and in common among the 3 main fibroblast subtypes. E) Violin plot representation of expression differences among fibroblast populations.

Fig 5. Regulation of ECs and SMCs by fibroblasts.

A) Angiopoietin-like factors 1 and 2 secreted by maintenance, secretory, and ANGPTL7⁺ fibroblasts regulate ITGA5-expressing ECs and SMC1 as predicted by CellChat ligand-receptor interactome analysis. Arrows inside bubbles indicate the direction of regulation, with the size of the incoming arrow representing the strength of the interaction. B) Expression levels of ANGPTL1, ANGPTL2, and the alpha (ITGA5) and beta (ITGB1) subunits of the integrin receptor in ECs, SMCs, and fibroblasts.

Fig 6. Immune cells in upper arm veins.

A) Focused UMAP of 4931 monocytes/macrophages (Mo/Mφ), 1165 T cells, 244 NK cells, 235 mast cells, and 142 neutrophils (Neu) isolated from upper arm veins. B) Localization of CD163⁺ monocytes/macrophages and CD8⁺ T cells by immunohistochemistry. Scale bars represent 100 μm. C-D) Feature plots indicating normalized expression level of markers in common (C) and different (D) between the two monocyte/macrophage subpopulations. E) Violin plot representation of expression differences among immune cell populations.

Fig 7. Ligand-receptor interactions among venous cell populations.

A) Global plot of outgoing (ligand) and incoming (receptor) interactions among cell populations in veins as predicted by CellChat. Venous ECs are the top receivers among all populations. Valvular ECs are the top senders of signals. Monocytes/macrophages (Mo/Mφ) are the main immune populations participating as senders and receivers of global interactions. B) Examples of pathways where immune cells act as senders (left) or receivers (right) of signals. C) Plot of outgoing and incoming interactions among cell populations specifically for the chemokine (CXCL/CCL) signaling pathway. Venous ECs are the main receivers of signals through the ACKR1 receptor, while IL1B⁺ Mo/Mφ are the top senders. D) Relative contribution of specific chemokines to the plot in C. Red boxes indicate chemokines secreted by IL1B⁺ Mo/Mφ, while the green box marks chemokines secreted by venous/capillary ECs (CCL14) or other cells including venous ECs (CCL2).