Skeletal muscle pericytes as signal mediators in the vessel wall

In this thesis a novel method to isolate and cultivate pericytes
from hamster skeletal muscle is presented. By choosing a two-step
approach with magnetic-bead cell sorting for the 3G5 antigen and
culture in a selective growth medium a high percentage of pericytes
could be reached and their nature could be verified in subsequent
characterization steps. Morphologically the cells had a typical
phenotype with flat and large cell bodies and long protruding
processes. Immunocytochemistry and western blotting could confirm
the expression of the previously reported pericyte markers NG2,
PDGFR-β, and αSMA. However, in accordance with previous reports,
even the cells isolated from the same muscle and tissue, were not a
uniform population. A certain heterogeneity was also present in the
functional studies investigating pericyte membrane channels by
electrophysiology. In these experiments in a subset of the pericyte
population the presence of the voltage sensitive potassium channel
KV1.5 could be demonstrated. Upon pharmacological stimulation of
these channels by FFA they were able to elicit a membrane
hyperpolarization and conduct it to neighboring endothelial cells
via gap junctions formed by connexin 43. The expression of the
KV1.5 channel could also be demonstrated by immunohistochemistry in
paraffin sections from hamster skeletal muscle. These findings are
consistent with the hypothesis that pericytes could act as signal
generators for hyperpolarization and vasodilatation in the vessel
wall. The coupling with endothelial cells might relay an electric
signal further along the vessel wall and lead to “EDHF mediated”
dilations [48]. Interestingly in tissue sections KV1.5 positive
pericytes were located predominantly at vascular branchings. We
additionally studied whether gap junctions may also form a pathway
for the exchange of miRNA between cells in a model co-culture
system using HeLa. For the detection of the miRNA a detector system
consisting of a luciferase reporter protein under control of a
miR124 sensitive 3’ UTR was established. Our experiments could not
conclusively confirm transfer of miRNA in this system. Similar
results were obtained with human miR15b. Taken together these
findings open up new possibilities for studying skeletal muscle
pericyte physiology and to test novel concepts integrating the
pericyte into the vascular signaling network under conditions of
health and disease.