TESTOSTERONE INCREASES

EXPRESSION OF ENDOTHELIAL ER-Β IN

NORMAL GLUCOSE BUT NOT IN HIGH
GLUCOSE ENVIRONMENT

I K H L A S M . J E N I E *, B U D I M U L Y O N O **, S O E D J O N O A S W I N **, S R I K A D A R S I H S O E J O N O **
* FACULTY OF MEDICINE & HEALTH SCIENCES, UMY
** FACULTY OF MEDICINE, NURSING & PUBLIC HEALTH, UGM
INTRODUCTION
• Endothelial cells are squamous epithelial cells, with
polygonal shape and mononucleus. Endothelial cells
make up the endothelial layer in the tunica intima of blood
vessels, heart chamber, lymphatic vessels, and serous
cavities of the body (Anonymous, 1994; De Caterina et
al., 2007).
• Iniatially, endothelial cells are considered to be passive,
but later it is known that it actively synthesizes, secretes,
and metabolizes various vasoactive substances that act in
autocrine, paracrine and endocrine manner to contribute
in maintaining vascular homeostasis (De Caterina et
al., 2007).
INTRODUCTION
• It has been recognized that endothelium plays important
roles in cardiovascular events. One of its roles is the
ability to synthesize and secrete tromboregulator factors,
such as NO and PGI2 , which would inhibit the activation
and aggregation of platelets (De Caterina et al., 2007).
• Recently, endothelial cells are considered as target
tissues of testosterone because they contains androgfen
receptors (AR), estrogen receptors (ER) and enzymes to
metabolize testosterone (Torres-Estay et al., 2015).
METABOLISM OF
TESTOSTERONE IN THE
ENDOTHELIAL CELLS
Animal lab & human:
• Androgen receptors and estrogen receptors (McGill &
Sheridan et al., 1981; Liu et al., 2005; Toth et al., 2008)
• 5α-reductase enzyme (Hatakeyama et al., 2002 cit. Norata et al.
2006)
• Aromatase enzyme (Milewich et al., 1987- Sasano et al., 1999-
Diano et al., 1999 cit Death et al., 2004; Nathan et al., 2001;
Mukherjee et al., 2002)
(Source: Liu, Death & Handelsman, 2003)
FURTHERMORE..
• Endothelium layer is a part of blood vessel walls facing to
the lumen, so at any time the luminal side of the
endothelial cells are exposed to blood, except the
capillaries that are not experiencing recruitment.
• Therefore, endothelial cells can be influenced by changes
in the blood, either chemical or mechanical, such as an
increase in the concentration of blood glucose, or
hyperglycemia, as occurs in patients with diabetes
mellitus.
PROBLEMS
In what extent does glucose environment influence the
metabolism of testosterone in the endothelial cells? Whether
glucose environment supports conversion of testosterone to
become estrogen in the endothelial cells, that in turn will
affect the endothelial estrogen receptors?
METHODS
2X4 FACTORIAL
DESIGN
Normal glucose High glucose
(5.6 mM) (22.4 mM)
T0 G1 G5

T 1 nM G2 G6

T 10 nM G3 G7

T 100 nM G4 G8
 SAMPLE

Primary culture
Isolation of
Collection of of human
endothelial cells
umbilical cord umbilical vein
with enzymatic
from midwifery endothelial cell
disaggregation
(HUVEC)
 TREATMENT

Monolayer Harvesting & Subculturing

subconfluency cell count (grouped)

Morphological &
immunological
identification
TREATMENT

Exposure of
Washed
testosterone
(treatment Stained &
& glucose
ended) & counting
medium for
fixated
24 hours
RESULTS

HUVEC at confluency (100x)

RESULTS
40

35

30

25

Endothelial cells 20
positively stained with NG
ER-β antibody
(%) 15 HG

10

0
T0 T1 T10 T100

-5
Group
RESULTS
Unpaired t-test:
• Percentage of endothelial cells positively stained with ER-
β antibody in normal glucose medium without
testosterone (1.7 ± 2.74%) was significantly lower than in
normal glucose medium with 1 nM testosterone (14.09
±4.4%; p = 0.0001), 10 nM testosterone (11.01 ± 5.36%; p =
0.014), and 100 nM testosterone (23.21 ± 10.52%; p = 0.009)
RESULTS
Unpaired t-test:
• Percentage of endothelial cells positively stained with ER-
β antibody in high glucose medium without testosterone
(11.11 ± 7.35%) did not significantly differ with those in
high glucose medium with 1 nM testosterone (4.14 ±4%; p
= 0.11), 10 nM testosterone (4.53 ± 2.82%; p = 0.119), and
100 nM testosterone (6.22 ± 2.85%; p = 0.222)
RESULTS
Oneway ANOVA:
• Exposure of testosterone in incremental doses in normal
glucose medium significantly influenced percentage of
endothelial cells positively stained with ER-β antibody (p =
0.001)
• Exposure of testosterone in incremental doses in normal
glucose medium did not significantly influence percentage
of endothelial cells positively stained with ER-β antibody
(p = 0.107)
RESULTS
Analysis of variance for 2 x 4 factorial design:
• There was main effect of glucose medium to percentage of
endothelial cells positively stained with ER-β antibody (p =
0.002).
• There was main effect of testosterone to percentage of
endothelial cells positively stained with ER-β antibody (p =
0.012).
• There was interaction between testosterone and glucose
medium to percentage of endothelial cells positively
stained with ER-β antibody (p = 0.0001).
DISCUSSION
• Endothelial cells derived from human umbilical cord and
placentae villi have abundant ER- but a few of ER- (Toth
et al., 2008; Su et al., 2011)

• During human trophoblast differentiation, the ER- is

associated with a less, and ER- with the more
differentiated state (Bukovsky et al., 2003)
DISCUSSION
• In this study, exposure of testosterone increases the
expression of ER- in endothelial cells derived from
human umbilical vein

• In rat’s endothelial cells obtained from aorta, exposure of

testosterone increases expression of aromatase enzyme,
density, ER- and the synthesis of estrogen (Villablance et
al., 2013)

• In human cerebral vascular endothelial cells, ER- is

positively correlated with 17𝛽-estradiol concentrations (Tu
& Jufri, 2013)
DISCUSSION
• In this study, there is interaction between exposure of
testosterone and glucose to the expression of endothelial
ERβ

• In animal model of diabetes mellitus, the decreased

activity of aromatase enzyme is observed (Burul-Bozkurt
et al., 2010)
CONCLUSION
Exposure of testosterone increases expression of ER- in
cultured human endothelial cells derived from umbilical vein,
which may be attenuated during high glucose exposure.
ALHAMDULILLAH,
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