FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 134

standard error for percent positive was very large in every dose group analyzed, and
good correlation was not observed among any two antigens making robust statistical
analysis problematic. In fact, VWF and CD31 staining, for instance, was virtually
mutually exclusive. Differential immunophenotyping results led to the hypothesis that
rat CECs/CEPs are a complexmixture that may include cells that are in different states
of maturation or differentiation, originate from different sites, and/or are of hema-
topoietic and nonhematopoietic origin. This agrees well with published data describ-
ing many differences in growth, morphology, structural organization, protein
expression, and function in endothelial cells from various sources throughout the
vasculature [18, 21].

6.6.3 Secondary Validation

Despite the nonuniformity in surface protein expression, a cell population identified
as CD36 þ and/or DiI-Ac-LDL þ did contain an endothelial-like population as
confirmed by gene expression analysis. CD36 þ /Lin and/or DiI-Ac-LDL þ /Lin
populations were isolated by cell sorting and gene expression analysis (TaqMan)
performed using a small panel of endothelial-related gene probes. This panel included
TEK (TIE2, CD202B), MCAM (CD146, MUC18), EDN1 (endothelin1, ET-1), VWF,
FLT1 (VEGFR-1), FLK1 (KDR, VEGFR-2), PECAM1 (PECAM-1, CD31), and
CDH5 (VE-Cadherin, CD144). Gene expression paralleled flow cytometry results in
that not all “endothelial-specific” genes were present and differential expression was
observed across animals (control only). Only EDN1 was consistently expressed in
sorted CEC/CEP fractions. Utility of probes was confirmed using rat heart homo-
genate. This supported the hypothesis of a complex mixture of cell types within the
CD36 þ /DiI-Ac-LDL þ population. As with protein expression, heterogeneity in
gene expression in endothelium has been reported and attributed to differences in
functional or differentiation status that, in some cases, can be modulated by drug
treatment [22, 23]. Heterogeneity in gene expression patterns has also been demon-
strated across endothelial subtypes from distinct regions of the heart [24-26].
In addition to confirmatory gene expression experiments, cell culture of normal rat
PBMC and sorted CD36 þ Lin and DiI-Ac-LDL þ /Lin cells was performed. First,
a modified human colony forming unit (CFU)-Hill assay [27] using rat PBMC was
successfully employed to confirm existence of CEP. Longer term culture resulted in
disappearance of colonies and emergence of spindle-shaped cells that stained
positively for CD31, von Willebrand factor, and rat endothelial antigen (RECA)-1
and negatively for smooth muscle -actin (SMA), an immunophenotype consistent

with mature endothelial cells. CFU-Hill assay confirmed existence of CEP in normal
rat blood but required ProNectinF, as opposed to traditional fibronectin, as sub-
strate [28]. The CFU-Hill assay begins with PBMC that are plated for 2 days, then
nonadherent cells are removed and replated for the actual colony-forming assessment.
This nonadherent fraction was examined by flow cytometry to confirm existence of
CD36 þ /Lin cells. In addition, sorted CD36 þ /Lin cells were placed in culture
directly, treating them as “nonadherent PBMC.” Although colonies formed as with
PBMC, longer term culture did not produce outgrowth of endothelial-like cells. This

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