220 Hewett
and therefore a lack of a given endothelial marker may not preclude the endot-
helial origin of isolates (2). It is often useful to demonstrate the absence of
markers, such as stress fibers staining with smooth muscle _-actin and the
intermediate filament protein, desmin, that are characteristic of smooth muscle
cells and pericytes (20), both potential endothelial culture contaminants.
3.2.1. Key Endothelial Cell Markers
Over recent years, more specific endothelial markers have emerged, such as
PECAM-1 (13), E-selectin (14), ICAM-2, and VE cadherin (5), and they have
increased the ease of endothelial characterization. Here we focus on von
Willebrand Factor (vWF), PECAM-1, and E-selectin, which we believe to be
useful for the rapid identification of ECs. For more detailed literature on these
endothelial markers there are several reviews that cover in depth the character-
ization of ECs (2,3,5,6,19).
vWF is only expressed at significant levels in ECs, platelets, megakaryo-
cytes, and syncytiotrophoblast. In ECs it is stored in the rod-shaped Weibel-
Palade bodies that produce the characteristic punctate perinuclear staining.
These organelles are present in human ECs isolated from large vessels (1,6)
but have been reported to be scarce or absent in capillary endothelium from
various species (2,4). However, typical granular perinuclear staining for vWF
has been reported in human kidney, dermis (9), synovium, lung, stomach,
decidua, heart, adipose tissue, and brain (8) microvessel ECs.
PECAM-1 is constitutively expressed on the surface of ECs (>10
6
molecules/
cell), and to a lesser extent in platelets, granulocytes, and a sub-population of
CD8+ lymphocytes (13). PECAM-1 staining of ECs in vitro is characterized
by typical intense membrane fluorescence at points of cell-cell contact (Fig. 2).
E-selectin expression appears to be unique to ECs (14). Although it is not
constitutively expressed by the majority of ECs, E-selectin expression is
induced following stimulation with inflammatory cytokines such as tumor
necrosis factor-_ (TNF_) or interleukin-1` (IL-1`) (14). Absent on unstim-
ulated controls, intense E-selectin expression is induced by pretreatment for
4 h with 10 ng/mL of TNF_ reaching a maximum at 4–8 h before returning to
background levels.
3.2.2. Immunocytofluoresent Characterization of Endothelial Cells
Immunocytoflourescence represents a simple rapid technique to character-
ize ECs. Outlined below is a staining protocol that we have routinely used for
this purpose.
1. Preparation of ECs on glass slides. Multiwell glass chamber slides are extremely
useful for this purpose as multiple tests can be performed on the same slide con-