A Guide to Endothelial Cell Markers

Endothelial cells line the interior surface of the circulatory system, forming a barrier that regulates the transport of fluids, substances, and cells to and from various tissues. This system includes blood vessels, which transport nutrients and oxygen to tissues throughout the body, and lymphatic vessels, which drain interstitial fluid; both serve as conduits for immune cell trafficking. Although most validated endothelial cell markers label blood vessel endothelial cells and lymphatic endothelial cells indiscriminately, some label lymphatic cells exclusively. Emerging datasets generated by next-generation sequencing have uncovered novel endothelial markers, enabling the identification of new endothelial subsets. However, whether these transcriptional markers are suitable for protein detection methods remains to be seen.

General Endothelial Markers

Pure endothelial cell cultures have several distinguishing features. In addition to their cobblestone morphology, they express Von Willebrand factor (VWF), a glycoprotein involved in blood clotting, and angiotensin-converting enzyme (ACE/CD143), a protease that generates angiotensin II. Endothelial cells also have specialized organelles called Weibel-Palade bodies, which store VWF alongside P-selectin (encoded by CD62P), which participates in leukocyte recruitment. Large multimers of VWF that form on endothelial surfaces are processed into shorter forms by the metalloproteinase ADAMTS13. Given their functional importance, VWF, ACE, P-selectin, and ADAMTS13 are key endothelial markers.

This diagram highlights general markers for endothelial cells and markers selective for lymphatic endothelial cells.

Many glycoproteins other than VWF decorate endothelial surfaces, with the vast majority serving as adhesion molecules — essentially membrane-bound tethers — for platelets, pericytes, and circulating immune cells. Cellular engagement of these tethers regulates a number of biological processes, ranging from blood clotting to leukocyte transmigration. During inflammation, pro-inflammatory cytokines such as TNF-alpha activate endothelial cells, inducing adhesion molecule expression and thereby enabling circulating leukocytes to attach to sites of vascular injury and inflammation. P-selectin (SELP) acts as a first point of contact for such leukocytes, with PECAM1, VCAM1, ICAM1, ICAM2, CD47, and E-selectin (SELE) acting as additional contact points for vascular adhesion. VE-cadherin (CDH5), NECTIN2, and ESAM localize to the junctions between endothelial cells, where they participate in leukocyte transmigration. Other adhesion molecules enable interactions between endothelial cells and platelets (ITGB3, CD151), pericytes (CD248, MCAM, ITGA4), smooth muscle cells (MCAM), erythrocytes (CXCL16) and cardiomyocytes (ITGB1), to provide a few examples.

Markers of endothelial maturation can also be used to help identify endothelial cells. Stemness marker CD34 and the structurally related podocalyxin (PODXL) protein appear on subsets of endothelial cells with stem-like properties. Adult endothelial progenitors also express CD44; these cells circulate, enabling them to easily migrate to sites of vessel formation (angiogenesis) and injury, where they can generate new endothelial cells.

Select proteins of angiogenic pathways, including the Notch-VEGF and angiopoietin-Tie signaling pathways, can also serve as markers. This list includes VEGFR2, NOTCH1, TIE1, and TIE2.

The protein products of ANPEP and ANTXR1 can aid the detection of endothelial tumors due to their expression in tumor vasculature.

Other endothelial markers include F3, which encodes the coagulation cofactor thromboplastin; the proinflammatory scavenger receptor CD36; the antiangiogenic receptor CD160; the cytokine receptors IL13RA1 and IL1R1; the costimulatory molecules CD80 and CD86; the TNF-α receptor TNFRSF1B; the tyrosine kinase receptor c-Kit; the coagulation inhibitors PROCR and thrombomodulin (THBD); the electrophile-glutathione conjugate transporter RALBP1; hyaluronan (HA) receptor homologs STAB1 and STAB2; and the tight junction molecule CLDN5.

Blood Vessel vs. Lymphatic Endothelial Markers

Most endothelial markers label endothelial cells of both blood and lymphatic vessels, but a few markers label only lymphatic endothelial cells.

One such marker, lymphatic vessel endothelial hyaluronan 1 (LYVE1), acts as a receptor for the extracellular matrix component hyaluronic acid (HA). Three more lymphatic endothelial cell markers participate in lymphangiogenesis. These markers include PROX1, a lymphatic-specific homeobox gene that controls the development of lymphatic progenitors, the PROX1 target gene podoplanin (PDPN), and vascular endothelial growth factor receptor-3 (VEGFR3/FLT4). All four of these markers can be used in immunohistochemical studies of the lymphatic endothelium.

Organ-Specific Endothelial Markers

Emerging omics data have provided a bevy of novel endothelial markers. The high-resolution of these technologies have even revealed markers of endothelial subsets, enabling the venous and arterial endothelial cells of specific organs to be distinguished by marker gene expression.

In one example, Munji and colleagues (2019) utilized RNA sequencing to identify differences between brain endothelial cells and so-called “peripheral” (i.e. nonbrain) endothelial cells. Brain endothelial cells preferentially expressed genes related to the Wnt-beta-catenin signaling pathway, namely Lef1, Fzd3, Notum, Apcdd1, Axin2, dixdc1 and Tnfrsf19. This is unsurprising given the role of this signaling pathway in brain-specific angiogenesis and blood-brain barrier formation and maintenance. By contrast, peripheral endothelial cells could be distinguished by their expression of several Hox genes, including Hoxa5 and Hoxb4. ALPL, which encodes tissue nonspecific alkaline phosphatase (TNAP) and is thought to play a role in the progression of neuronal loss in Alzheimer’s disease, also specifically labels brain endothelial cells. These genes are just a few markers that are differentially expressed by brain and peripheral endothelial cells.

Similar datasets have been generated for endothelial cells of other organs, too. For a more complete summary of organ-specific endothelial markers, refer to Trimm and Red-Horse (2023).

Table of Endothelial Cell Markers

The table below lists characteristic endothelial cell markers as described by review literature. The list includes a variety of marker types, including mostly cell surface proteins but also transcription factors, enzymes, signaling proteins, and structural proteins. Accompanying each marker are links to relevant antibodies and ELISA kits that can be used to detect endothelial cells in vitro and in vivo. The associated products are offered by a variety of manufacturers and can serve as a useful reference for endothelial cell characterization.

Note: Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

References

1. Ordóñez NG. Immunohistochemical endothelial markers: a review. Adv Anat Pathol. 2012;19(5):281-295. doi:10.1097/PAP.0b013e3182691c2a

2. Goncharov NV, Nadeev AD, Jenkins RO, Avdonin PV. Markers and Biomarkers of Endothelium: When Something Is Rotten in the State. Oxid Med Cell Longev. 2017;2017:9759735. doi:10.1155/2017/9759735

3. Rakocevic J, Orlic D, Mitrovic-Ajtic O, et al. Endothelial cell markers from clinician's perspective.Exp Mol Pathol. 2017;102(2):303-313. doi:10.1016/j.yexmp.2017.02.005

4. Munji RN, Soung AL, Weiner GA, et al. Profiling the mouse brain endothelial transcriptome in health and disease models reveals a core blood-brain barrier dysfunction module.Nat Neurosci. 2019;22(11):1892-1902. doi:10.1038/s41593-019-0497-x

5. Goncharov NV, Popova PI, Avdonin PP, et al. Markers of Endothelial Cells in Normal and Pathological Conditions.Biochem (Mosc) Suppl Ser A Membr Cell Biol. 2020;14(3):167-183. doi:10.1134/S1990747819030140

6. Trimm E, Red-Horse K. Vascular endothelial cell development and diversity.Nat Rev Cardiol. 2023;20(3):197-210. doi:10.1038/s41569-022-00770-1