Differences in Mitochondrial Activity between Active and Inactive
Hepatic Stellate Cells
Department of Interventional Radiology, Johns Hopkins University School of Medicine
Abstract
Liver fibrosis is one of the main causes of death by liver disease
in the United States caused by severe scarring of the internal tissue of
the liver by extracellular matrix (ECM) producing hepatic stellate cells
(HSCs). However, a suitable clinical drug has not yet been developed
due to the lack of understanding on how to induce HSCs into an
inactive state.
In this study, the mitochondria were pinpointed as the most likely
cause of creating an activated state in the HSCs since they provide the
cell with energy. The MitoTracker Green fluorescent stain and the
Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) fluorescent
stain were used to qualitatively determine a difference in the activity in
the mitochondria and mitochondrial membrane potential, respectively,
between active and inactive HSCs. In addition to a qualitative analysis,
a glucose uptake study, a lactate analysis, and a ATP quantification
study were done to ensure the validity of the staining results. The study
found that there is a higher mitochondrial membrane potential in active
HSCs which was supported by the findings of the quantitative data, so
a drug can be developed to lower the mitochondrial membrane
potential of fibrotic livers to induce an HSC into an inactive state and
reduce the scarring of the liver.
Introduction
Almost 2% of the total deaths in the United States were caused
by liver disease or cirrhosis in 2014 resulting in the loss of over
38,000 lives, up 2% from the previous year. One of the main causes of
liver disease is liver fibrosis which can lead to cirrhosis and
hepatocellular carcinoma. It is caused by the buildup of excessive scar
tissue that cannot be broken down in an efficient manner. The onset of
liver fibrosis is caused mostly by chronic infection with hepatitis C or
hepatitis B virus (HCV or HBV), heavy alcohol consumption, toxins,
trauma, or a genetic predisposition.
The liver is composed of four main types of cells: Hepatocytes,
Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells.
When the liver is injured, hepatic stellate cells activate and repair the
injured tissue and in the process, they produce extracellular matrix
(ECM). However, after repeated injury, the hepatic stellate cells
remain in a constant activated form and secrete an excessive amount
of ECM. These ECM proteins can create thread-like structures leading
to the scarring of liver tissue which can result in liver fibrosis and, if
left untreated, to cirrhosis and hepatocellular carcinoma.
The purpose of this project is to determine if there is a
qualitative or quantitative difference between an active and inactive
hepatic stellate cells. If so, liver fibrosis can be detected early and
drugs can be used to induce an inactive state of the hepatic stellate
cells. This would lead to a decrease in cases of cirrhosis and hepatic
carcinoma.
Methods
Cell Plating-
Rat HSCs and LX2 cells (human cell line) were plated on 2, 8-
chamber slides. Four of the chambers were coated with Matrigel with
reduced growth formula to induce an inactive state. 10000 LX2 cells
were plated in each chamber on one slide and 10000 rat HSCs were
plated in each chamber on the second slide.
Priya Gajendiran, Shanm Ganapathy Ph.D.
Graphs and Figures
Graph 1 shows the ATP Quantification of 10000
LX2 Active cells, LX2 Inactive cells, Rat HSC
Active cells, and Rat HSC Inactive cells. The ATP
Quantification was done in 96-well plates using the
ELISA method.
Figure 1 The top images are of Active LX2 cells
and the bottom images are of Inactive LX2 cells
each with a MitoTracker Green stain. This image
was taken using a 3i Spinning Disc Confocal
Microscope at 63x oil.
Graph 2 shows the 3H-2-deoxyglucose uptake of
10000 LX2 Active cells, LX2 Inactive cells, Rat
HSC Active cells, and Rat HSC Inactive cells. The
glucose uptake analysis was done using a kit.
Figure 3 The top images are of Active LX2 cells
(left with DAPI, right is without) and the bottom
Graph 3 shows the Lactate production of 10000
images are of Inactive LX2 cells (left with
DAPI, right is without) each with a TMRM
LX2 Active cells, LX2 Inactive cells, Rat HSC
Active cells, and Rat HSC Inactive cells. The
stain. This image was taken using a 3i Spinning
Lactate production analysis was done in 96-well
Disc Confocal Microscope at 63x oil.
plates using a kit.
Figure 2 The top images are of Active Rat
HSCs and the bottom images are of Inactive
Rat HSCs each with a MitoTracker Green
stain. This image was taken using a 3i Spinning
Disc Confocal Microscope at 63x oil.
Figure 4 The top images are of Active Rat
HSCs (left with DAPI, right is without) and the
bottom images are of Inactive Rat HSCs (left
with DAPI, right is without) each with a
TMRM stain. This image was taken using a 3i
Spinning Disc Confocal Microscope at 63x oil.
Cell Staining-
Manufacturer protocols were followed for the TMRM,
MitoTracker, and DAPI stains. (See below for more in depth
protocols.)
Cell Imaging-
The fluorescent stains were analyzed by imaging cells with a
spinning 3i confocal microscope. After taking images with the
microscope, the images were stacked and analyzed further using
ImageJ and Adobe Photoshop.
Quantitative Studies-
The ATP analysis will be done using the ELISA method and the
Lactic Acid and glucose assay will be done using a kit. (See
below for more in depth protocols.)
Results and Discussion
MitoTracker Stain
The MitoTracker fluorescent stain was more intense and brighter in the
Active cell lines than the Inactive cell lines. This supported the current idea
since and active cell would use more energy and would therefore have
more active mitochondria.
TMRM Stain
The TMRM stain, which fluoresces more intensely in the presence of a
high mitochondrial membrane potential, also yielded different brightness
and intensity between the active and inactive cell lines. It was found that an
active cell tended to have mitochondria with higher membrane potentials
than an inactive cell.
Glucose Uptake Study
The results of the glucose study showed that active cell lines took up more
glucose than inactive cell lines. This was to be expected since active cells
would be more likely to undergo aerobic respiration which would result in
the mitochondria absorbing more glucose to use in cellular respiration than
an inactive cell.
ATP Quantification
The results of the ATP Quantification showed that active cell lines took up
more glucose than inactive cell lines. This was to be expected since active
cells would be more likely to undergo aerobic respiration which would
result in the mitochondria going through glycolysis, the Krebs cycle (Citric
Acid cycle), and oxidative phosphorylation which would result in
significantly more ATP than an inactive cell which undergoes anaerobic
respiration.
Lactate Production
The results of the Lactate analysis showed that inactive cell lines produced
more lactate than active cell lines. This was to be expected since inactive
cells would be more likely to undergo anaerobic respiration which would
result in the mitochondria going through glycolysis and then fermentation
resulting in the formation of lactate whereas an active cell would undergo
aerobic respiration.
Future Studies
A quantifiable difference in the mitochondrial activity of an active
and inactive HSC was found though the data collected in this study.
A significant difference in the mitochondrial membrane potential of
active and inactive HSCs indicates that mitochondria-targeted drugs
can be used to decrease the secretion of ECM, so the next step of
this study is to test various drugs that could lower the mitochondrial
membrane potential.
Acknowledgements
Dr. Shanm Ganapathy from the Department of Interventional
Radiology at Johns Hopkins Hospital
Ms. Loza Lee, Ms. Barbara Smith, and Mr. Hoku West-Foyle
of JHH MicFac