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Ultrastructure Changes of Cardiac Lymphatics During Cardiac Fibrosis in Hypertensive Rats.

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THE ANATOMICAL RECORD 292:1612–1618 (2009)
Ultrastructure Changes of Cardiac
Lymphatics During Cardiac Fibrosis in
Hypertensive Rats
XIAODONG LI,1 TATSUO SHIMADA,2 YAFANG ZHANG,1 XIANLI ZHOU,3
1
AND LINGHUI ZHAO *
1
Department of Anatomy, Harbin Medical University, Harbin, China
2
Department of Health Science, Faculty of Medicine, Oita University, Oita, Japan
3
Ultrasonic Department, The Second Clinical Hospital, Harbin, China
ABSTRACT
Hypertension is one of the most common diseases that induce a series
of pathological changes in different organs of the human body, especially
in the heart. There is a wealth of evidence about blood vessels in hypertensive myocardium, but little is known about structural changes in the
cardiac lymphatic system. To clarify the changes in structure of the cardiac lymphatic system during hypertension, we developed a hypertension
animal model with Dahl S rats and we used Dahl R rats as the control
group. We examined the expression of collagen fibers, atrial natriuretic
peptide, connexin43, and LYVE-1 in the rat heart by immunohistochemistry. The ultrastructure of the cardiac lymphatics was detected by transmission electron microscopy and scanning electron microscopy. We
demonstrated extensive lymphatic fibrosis in the hearts of the Dahl S
hypertension group, characterized by increased thin collagen fibrils that
connected with the lymphatics directly. Ultrastructural changes in the
cardiac lymphatic endothelium such as an increase of vesicles and occurrence of vacuoles, active exocytosis, and cytoplasmic processes, restored
the draining of tissue fluid. Our study suggests that during hypertension,
the changes in structure of the cardiac lymphatics may be one important
factor involved in cardiac fibrosis. Therefore, the lymphatics may be a
possible target for reducing fibrosis in the treatment of hypertension.
C 2009 Wiley-Liss, Inc.
Anat Rec, 292:1612–1618, 2009. V
Key words: hypertension; Dahl S; lymphatic; fibrosis
INTRODUCTION
The lymphatic system is widely distributed in the heart.
It originates from the lymphatic capillaries in the subendocardium, passes through the lymphatics in the myocardium and collecting lymphatics in the subepicardium, and
eventually forms the lymphatic trunks and joins the venous circulation (Shimada et al., 1990). Several lines of evidence have demonstrated that mechanical or reactive
obstruction of the cardiac lymphatics causes pathological
changes in the myocardium, endocardium, cardiac valves,
and conduction system (Gloviczki et al., 1983).
Myocardial fibrosis is a significant primary change
resulting from hypertension (Cooper et al., 1990; Koren
et al., 1991; Brilla et al., 1993). Myocardial fibrosis involves
deposition of dense collagen fibers among the myocardium
and around the cardiac blood vessels, and results in subseC 2009 WILEY-LISS, INC.
V
quent symptoms including eventual heart failure. The
mechanism for myocardial fibrosis is still controversial.
However, there is now a wealth of evidence suggesting that
lymphedema may be one important factor that is closely
related to myocardial fibrosis (Laine and Allen, 1991; Stolyarov et al., 2002; Kong et al., 2006). Lymphedema results
in the increase of interstitial pressure (Laine and Allen,
1991) and eventually causes synthesis and accretion of
*Correspondence to: Linghui Zhao, Department of Anatomy,
Harbin Medical University, Harbin, China.
E-mail: zhao-lh@163.com
Received 18 January 2009; Accepted 7 May 2009
DOI 10.1002/ar.20943
Published online 15 August 2009 in Wiley InterScience (www.
interscience.wiley.com).
ULTRASTRUCTURE CHANGES OF CARDIAC LYMPHATICS
collagen fibers. TGF-b1 may be involved in this process
(Lijnen et al., 2000; Kuwahara et al., 2002). Lymphedema
is closely associated with the function of the cardiac lymphatics. However, though hypertensive cardiac blood vessels have been well studied, little is known about the
relationship between the cardiac lymphatics and myocardial fibrosis during the condition of hypertension.
This study was undertaken to investigate the structural changes in the cardiac lymphatics during hypertensive fibrosis. Currently, there are many different
types of animal models for hypertension; we chose saltsensitive rats as follows: Dahl S rats as the experimental
group and salt-resistant rats as follows: Dahl R rats as
the control group. The hypertension animal model was
developed by feeding the Dahl S rats with a high salt
diet, and structural changes in the cardiac lymphatics
under conditions of hypertension were investigated. A
better understanding of the cardiac lymphatics will
hopefully lead to treatment of myocardial fibrosis.
MATERIALS AND METHODS
Hypertension Animal Model
Ten Dahl S rats and 10 Dahl R rats, male, 8 weeksold, bought from the Jiudong Company, were fed on 8%
NaCl diets for 1 month. Systolic blood pressure in the
tail artery was measured every week by the tail cuff
method. After the rats were sacrificed under deep anesthesia with an injection of sodium pentobarbital (50 mg/
kg), heart perfusion with 0.9% buffered saline solution
was carried out under 103–120 mmHg from the apex of
heart, the hearts were then removed.
Sample Preparation
For light microscopy observation, two samples of both
atriums and ventricles of every rat were obtained horizontally and transmurally, in total 40 blocks were fixed in
4% paraformaldehyde to prepare tissue sections of 4 lm
thickness. For transmission electron microscopy (TEM)
observation, section of both the atriums and ventricles of
every rat were obtained horizontally and divided into
small blocks (1 mm 1 mm 2 mm), samples were all
immersed in 2% glutaraldehyde solution at 4 C. For scanning electron microscopy (SEM) observation, two samples
of both atriums and ventricles of every rat were obtained
horizontally and transmurally, in total 40 blocks were
immersed in 2% glutaraldehyde solution at 4 C.
Van Gieson Staining
Paraffin sections (4 lm) were deparaffinized in dimethylbenzene, rehydrated through a graded alcohol series,
spent 15 min in Weigert’s iron hematoxylin and 3 min in
Van Gieson’s solution, were counterstained in hematoxylin, dehydrated, and mounted.
Immunohistochemistry
Sections were treated with 0.3% H2O2 in PBS for
15 min to inhibit intrinsic peroxidase activity and 10%
blocking solution for 20 min to prevent nonspecific antibody binding. They were then incubated overnight at
4 C with either rabbit anti rat atrial natriuretic peptide
(ANP) polyclonal antibody (diluted 1:100; YII-Y330-EX,
purchased from Cosmo Bio), rabbit anti rat connexin43
1613
polyclonal antibody (diluted 1:100; ab66151, purchased
from Abcam), or rabbit anti rat LYVE-1 monoclonal antibody (diluted 1:200; sc-80170, purchased from Santa
Cruz Bio). After rinsing in PBS, slides were then incubated for 25 min at room temperature with polyHRP
goat anti-rabbit IgG. Slides were rinsed again with PBS
and were stained with DAB, counterstained in hematoxylin, dehydrated, and mounted. Control immunostaining
was carried out by the same procedure in which the first
antibody was replaced by nonimmunized serum.
Transmission Electron Microscopy
Specimens were postfixed for 30 min at room temperature in 1% OSO4, and dehydrated in a graded series of
ethanol and embedded in epoxy resin. Semi-thin sections
(1.0 lm thick) were stained with 1% toluidine blue for
light microscopy. In different rats, the numbers of blocks
containing lymphatics were not same, to insure the
selection bias minimized, 6 blocks of ventricle containing
lymphatic and 4 blocks of atrium containing lymphatic
from every rat were chose randomly, in total 200 blocks
were prepared for ultra-thin slice, ultra-thin sections
(80–100 nm thick) were stained with uranyl acetate and
lead citrate and examined under TEM.
Scanning Electron Microscopy
Specimens were further treated with 1% Tween for
3 hr at 37 C and were then washed thoroughly in
distilled water, immersed in a 1% aqueous solution of
tannic acid for 2 hr, postfixed in cacodylate buffered 1%
osmium tetroxide for 2 hr, dehydrated in graded concentrations of ethanol, and then dried by means of the
t-butyl alcohol freeze-drying method. The specimens
were then sputter-coated with gold and examined in an
H-800 scanning electron microscope.
Statistical Analysis
The results of Van Gieson staining and immunohistochemical staining were examined by a professional pathological doctor using the Image-Pro Plus 5.0 image
analyze system. Five views on every slides were chosen
randomly and the area density of positive expression
(positive area/total area) was quantified. Twenty views of
TEM with the same magnification were randomly chosen
from the Dahl S and Dahl R groups. The total numbers,
area density (lm2/lm2), and number density (N/lm2) of
uncoated vesicles in lymphatic endothelium were analyzed. All of the data are presented as mean SD and
were analyzed by SPSS 10.0 statistical analysis software.
A value of P < 0.05 indicates statistical significance.
RESULTS
Hypertension Animal Model
After the rats were fed on 8% NaCl diets for 1 month,
the systolic blood pressure of rats was measured, the
maximum, minimum, and mean systolic blood pressure
in the Dahl S group were 244, 228, and 232 mmHg,
respectively. The maximum, minimum, and mean systolic
blood pressure in the Dahl R group were 135, 121, and
126 mmHg, respectively. The Dahl S rats had higher
blood pressure than Dahl R rats; over 180 mmHg is
regarded as the hypertension standard in rats by most
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LI ET AL.
Fig. 2. The area density of collagen fibers in the Dahl S group is
significantly higher than that of the Dahl R group. The area density of
ANP in the atrium of the Dahl S group was significantly higher than
that of the Dahl R group. The area density of connexin43 in the Dahl S
group was significantly lower than that in the Dahl R group.
Fig. 1. a: Dahl R; sparse collagen fibers (arrow) are seen around
the artery (200). b: Dahl S; a thick layer of collagen fibers are distributed around the artery and among the myofibrils (arrow) (200).
c: Dahl R; immunoreactive particles of ANP are found near the nucleus of myofibrils in the atrium. The arrow indicates the epicardium of
the atrium (400). d: Dahl S; immunoreactive particles of ANP are
seen in the cytoplasm of myofibrils in the atrium. The arrow indicates
the epicardium of the atrium (400). e: Dahl R; a large amount of connexin43 immunoreactive particles can be seen between the adjacent
myofibrils in the ventricle (400). f: Dahl S; immunoreactive particles
of connexin43 were decreased in number in the ventricle (400).
researchers (Dal Canton et al., 1989). Therefore, Dahl S
rats were designated as the hypertension group, and
Dahl R rats were the control group.
Myocardial Fibrosis
Using Van Gieson staining, the myofibrils appeared
yellow and the collagen fibers were red. Rare collagen
fibers were distributed around the blood vessels and
among myofibrils in the Dahl R group (Fig. 1a). In the
Dahl S group, the collagen fibers were increased in the
myocardium, especially around the coronary artery
where the collagen fibers became dense and thick. In
addition, dense collagen fibers were deposited among the
myofibrils in a cord shape (Fig. 1b). The area density of
collagen fibers in the Dahl S group (0.193 0.012) was
significantly higher than that in the Dahl R group
(0.122 0.007, Fig. 2), P < 0.05.
Expression of atrial natriuretic peptide
in the Atrium
Expression of ANP was observed in the cytoplasm of
myofibrils in the atrium but not the ventricle in the two
groups. In the Dahl R group, the immunoreactive par-
Fig. 3. Immunohistochemical staining of LYVE-1. a: Dahl S; arrow
indicates one enlarged lymphatic vessel, round in shape, near the coronary artery (B) in the ventricle (400). b: Dahl S; a dilated lymphatic
vessel (arrow) in the subepicardium in the ventricle showed an oval
shape (200). c: Dahl S; dilated lymphatic vessels (arrow) among the
myocardium in the ventricle (400). d: Dahl R; a lymphatic vessel
(arrow) with an irregular shape near artery (B) in the ventricle (400).
e: Dahl R; one lymphatic vessel (arrow) with an irregular lumen in the
subepicardium in the ventricle (400). f: Dahl R; lymphatic vessels
(arrow) with narrow lumen among the myocardium in the ventricle
(400).
ticles were mainly near the nucleus of myofibrils
(Fig. 1c). In contrast, the immunoreactive particles in
the Dahl S group were found in the whole cytoplasm of
myofibrils and showed a patch-like shape (Fig. 1d). The
area density of expression of ANP in the Dahl S group
(0.121 0.007) was higher than that in the Dahl R
group (0.049 0.032, Fig. 2), P < 0.05.
Expression of Connexin43 in the
Intercalated Disk
Immunoreactive particles of connexin43 appeared
brown color and were in the intercalated disks of adjacent
myofibrils (Fig. 1e,f). In the Dahl S group, the immunoreactive particles were decreased in number and were
absent in some myofibril borders when compared with
the Dahl R group. The area density of expression of ANP
1615
ULTRASTRUCTURE CHANGES OF CARDIAC LYMPHATICS
TABLE 1. Uncoated vesicles in lymphatic
endothelium of Dahl(S) and Dahl(R) rats
Number of Number of
views
vesicles
Dahl (S)
Dahl (R)
P value
20
20
686
426
<0.05
Area
density
(lm2/lm2)
Number
density
(N/lm2)
0.34 0.05 41.42 2.64
0.16 0.06 17.67 3.02
<0.05
<0.05
irregular shape and were represented by a narrow
lumen (Fig. 3d–f).
Ultrastructure of the Cardiac Lymphatics
Fig. 4. a: Dahl S; dilated collecting lymphatic vessel in the ventricle.
The arrow indicates smooth muscle of the collecting lymphatic vessel,
bar: 5 lm, TEM. b: High magnification of Fig. 4a. The arrow indicates
vacuoles with a large diameter that appeared in lymphatic endothelial
cells, bar: 400 nm, TEM. c: Dahl S; a lymphatic capillary in the atrium.
The arrows indicate vacuoles with a large diameter in the endothelium,
bar: 500 nm, TEM. d: Dahl S; a lymphatic capillary in the ventricle was
rich in uncoated vesicles (white arrow), black arrow indicates a
expanded rough surfaced endoplasmic reticulum, bar: 500 nm, TEM.
e: Dahl S; a lymphatic capillary in the ventricle; black arrow indicates
a vacuole with a large diameter, white arrow shows a damaged mitochondrion, bar: 500 nm, TEM. f: Dahl S; a lymphatic capillary in the
atrium; arrow indicates a uncoated vesicle, bar: 500 nm, TEM.
in Dahl S group (0.0045 0.087) was lower than that in
the Dahl R group (0.0243 0.042, Fig. 2), P < 0.05.
Distribution and Shape of the
Cardiac Lymphatics
LYVE-1 is a special type of marker for the lymphatics.
The cardiac lymphatic wall stains brown with LYVE-1
antibody. We found that blood vessels showed negative
results for LYVE-1. The lymphatics are distributed in
every layer of the rat heart, including the subendocardium, myocardium, and subepicardium. Most of the lymphatics in the Dahl S group lost their normal shape and
were dilated (Fig. 3a–c), especially those near the coronary arteries, which always showed a round shape. The
lymphatics in the Dahl R group still maintained an
Using TEM, damage of cellular organelles such as the
mitochondrion could be observed in the endothelium of
lymphatic capillaries and collecting lymphatics in the
Dahl S group (Fig. 4d,e). Two types of vesicles were present in both of the two groups; uncoated vesicles and
coated vesicles. Coated vesicles were infrequently seen
in the two groups. In contrast, plenty of uncoated
vesicles with different diameters were found in the endothelial cytoplasm in the Dahl S group. Using 20 views
with same magnification in the two groups, the number,
area density, and number density of uncoated vesicles in
the Dahl S group were higher than those in the Dahl R
group (Table 1). In addition to the coated and uncoated
vesicles, vacuoles with larger diameter occurred only in
the collecting lymphatics (Fig. 4a,b) and lymphatic capillary (Fig. 4c) in Dahl(S) group, there were more
uncoated vesicles in the lymphatic endothelium in the
Dahl S group (Fig. 4f) than in the Dahl R group.
Increased fibroblast and collagen fibers were
assembled near the lymphatics in the Dahl S group (Fig.
5a), and in some cases, collagen fibrils were connected
with the lymphatic endothelium directly (Fig. 5b). With
regard to collagen fibers around the lymphatics, the
Dahl S group mostly had thin collagen fibrils with a diameter of 30–40 nm (Fig. 5c) and the Dahl R group
had thick collagen fibrils with a diameter of 50–60 nm
(Fig. 5d). In the Dahl S group, thin threads could be
seen protruding from the collagen fibrils (Fig. 5e). In
contrast, the border of collagen fibrils in the Dahl R
group were smooth (Fig. 5f).
Active exocytosis to the lymphatic lumen (Fig. 6a) and
a large amount of cytoplasmic processes towards the
lymphatic lumen (Fig. 6b) could also be seen in the lymphatic endothelium in the Dahl S group. Anchoring filaments lay around the lymphatics and connected with
the endothelium and collagen fibers in the Dahl S group,
and damage in certain parts of the lymphatic endothelium was observed (Fig. 6c). There were mainly three
types of intercellular junctions in the lymphatic endothelium in the two groups, including the end-to-end type,
overlapping type, and interdigitating type. However,
open type intercellular junctions were rarely seen and
there was no difference in the number of these junctions
between the two groups. Using SEM, similar results to
the TEM were obtained. In the Dahl S group, lots of collagen fibrils that connected with the lymphatic endothelium directly and cytoplasmic processes towards the
lumen were observed (Fig. 7a,b) when compared with
sparse collagen fibrils around the lymphatics in the Dahl
R group (Fig. 7c,d).
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LI ET AL.
Fig. 5. a: Dahl S; lymphatic vessel in the ventricle is surrounded by
lots of fibroblasts (arrow), L: lymphatic vessel, bar: 5 lm, TEM.
b: Dahl S; lymphatic vessel in the ventricle is rich in uncoated vesicles
(black arrow) and surrounded by abundant collagen fibers (white
arrow). J indicates an overlapping junction of endothelial cells, bar:
500 nm, TEM. c: Dahl S; lymphatic vessel in the ventricle is surrounded by thin collagen fibrils (white arrow). The ring shows one
bunch of collagen fibers and the black arrow indicates an overlapping
intercellular junction, bar: 400 nm, TEM. d: Dahl R; lymphatic vessel in
the ventricle is surrounded by thick collagen fibrils (white arrow). The
ring shows two bunches of collagen fibers. The black arrow indicates
one end-to-end type intercellular junction, bar: 400 nm, TEM. e: Dahl
S; thin threads can be seen protruding from the collagen fibrils, bar:
150 nm, TEM. f: Dahl R; the border of the collagen fibrils was smooth,
bar: 150 nm, TEM.
Fig. 6. a: Dahl S; lymphatic vessel in the ventricle is rich in
vacuoles (white arrow) and anchoring filaments (F), and active exocytosis can be seen on the luminal surface (black arrow). C indicates
collagen fibers, M indicates myofibrils, bar: 500 nm, TEM. b: Dahl S;
lymphatic vessel in the ventricle, vacuoles (white arrow) and several
cytoplasmic processes (black arrow) towards the lumen are present,
C indicates collagen fibers, bar: 500 nm, TEM. c: Dahl S; the arrow
shows rupture of the lymphatic endothelium, bar: 400 nm, TEM.
Fig. 7. a: Dahl S; the arrow shows one intercellular junction of the
interdigitating type in lymphatic endothelial cells (L), bar: 3 lm, SEM.
b: Dahl S; the short arrow indicates dense collagen fibrils that are
connected with the lymphatic endothelium (L) directly. In addition, several cytoplasmic processes (long arrow) towards the lumen are present. Bar: 1.5 lm, SEM. c: Dahl R; the arrow indicates a nucleus in a
lymphatic endothelial cell. M indicates myofibrils, bar: 10 lm, SEM. d:
Dahl R; high magnification of c, arrow indicates a few collagen fibers
surrounding the lymphatic endothelium (L), M indicates myofibrils, bar:
1.5 lm, SEM.
DISCUSSION
Cardiac Lymphatic Fibrosis in the
Hypertensive Heart
Extensive cardiac fibrosis was found in the myocardium in the hypertensive group. Myocardial fibrosis is
one type of significant primary changes that result from
hypertension (Cooper et al., 1990; Koren et al., 1991;
Brilla et al., 1993). The extracellular matrix of the heart
is mostly composed of collagen fibers, and they consist of
mainly collagen type I and type III (Souza, 2002). Collagen fibers function to form the framework of the heart
and resist tensile force. Under conditions of hypertension, the synthesis of collagen fibers increases and they
are deposited among the myocardium and around the
blood vessels. The reason for cardiac fibrosis remains to
be determined, but lymphedema may be one important
factor that is closely related to myocardial fibrosis (Laine
and Allen, 1991; Stolyarov et al., 2002; Kong et al.,
2006). Cardiac fibrosis makes the heart ‘‘harder’’ and
has a negative influence on systole and diastole of the
heart (Wu et al., 2004). Little is known about the effect
of fibrosis on the cardiac lymphatics. However, there
ULTRASTRUCTURE CHANGES OF CARDIAC LYMPHATICS
have been some reports about its effect on other organs
(Yamauchi et al., 1998; Herd-Smith et al., 2001; Ji et al.,
2004). Our study demonstrated that the cardiac lymphatics in the Dahl (S) group also presented with fibrosis in the ventricle and atrium, which was similar to
that around blood vessels. Increased fibroblasts and collagen fibers were found around the lymphatics in the
Dahl S group. Moreover, the collagen fibers were mainly
composed of thin collagen fibrils, in contrast to thick collagen fibrils in the Dahl R group. Thin threads protruded from the collagen fibrils in the Dahl S group,
when compared with the smooth border of collagen
fibrils in the Dahl R group. This suggests that hypertension also induces the fibrillogenesis around the cardiac
lymphatics as well as in the blood vessels in the Dahl S
group, it was in agreement with other researchers’s
reports that fibrillogenesis was marked by the collagen
fibrils with increasing diameter (Rentz et al., 2007).
In normal tissue, only a few collagen fibrils are closely
associated with the lymphatic endothelium. For the
most part, they are separated from the lymphatic endothelium by anchoring filaments. However, TEM and
SEM demonstrated that in the Dahl S group numerous
collagen fibrils were connected with the abluminal surface of the lymphatics directly. When compared with
anchoring filaments, collagen fibrils represent more durability. Therefore, the collagen fibrils that connect with
the lymphatics may be more available on maintaining
the framework of lymphatics. The lymphatics in the
Dahl R group still appeared irregular in shape and had
a narrow lumen in the myocardium. However, the lymphatics in the Dahl S group were always enlarged in a
round or oval shape, especially those near the arteries.
The reason for this finding may be due to the decrease
in drainage of the lymph. On the other hand, it may be
due to numerous collagen fibers that connected with the
lymphatic endothelium, which would resulted in resistance to the gradually elevated interstitial fluid pressure.
Because myocardial fibrosis was more intense near the
arteries, the lymphatics near the arteries showed a
round shape more frequently than other parts in
cardium.
Structural Changes of the Cardiac Lymphatics
in the Hypertensive Heart
Lymphatic intercellular open junction in which adjacent cells do not come closer than 30 nm to each other is
more frequent in pathological conditions (Qu et al.,
2003). An increase in open junctions is seen in the small
intestinal lymphatics during thoracic duct blockage (Ji
and Kato, 2001). However, we found no trend in the
Dahl S group for an increase of open junctions. Because
of the increased intraluminal pressure, the tissue fluid
pushed the intercellular junction open, and this process
has always been considered to be involved in anchoring
filaments (Tammela et al., 2005). On the other hand, the
anchoring filaments play a more important role in closing the opened intercellular junctions. The anchoring filaments function like chordae tendineae in the cardiac
valve and insure that tissue fluid flows into the lymphatics in only one direction (Tortora and Nielsen,
2005). We found that numerous collagen fibrils were
directly connected with lymphatic endothelium in the
hypertensive group. As the anchoring filaments, these
1617
collagen fibrils could enhance limiting the amount of
open junctions even in high interstitial fluid pressure,
this may explain why open junctions were absent in the
Dahl S group.
The lymphatic endothelium contains endocytotic
vesicles of both the coated and uncoated types. Uncoated
vesicles are associated with transport across the lymphatic endothelium (P’Morchoe et al., 1984). Our study
investigated the number, number density and area density of uncoated vesicles in Dahl S and Dahl R rats, and
found that uncoated vesicles were increased in both
number and area in the Dahl S group. We speculate that
the increase in uncoated vesicles would restore the flow
of tissue fluid to the lymphatics. We also observed
another type of large vesicle or vacuole that was different from the above mentioned vesicles. Vacuoles
occurred in both the lymphatic capillaries and the collecting lymphatics in the Dahl S group. Large vesicles
were also found in the endothelial cytoplasm of a
blocked thoracic duct (Ji and Kato, 2001) and were
shown to promote the transport of lymph. It is unclear
whether such vacuoles elevate the efficiency of transportation across the lymphatic endothelium in hypertensive
cardium.
We observed an active exocytosis phenomenon in the
lymphatic endothelium in the Dahl S group and numerous cytoplasmic processes towards the lymphatic lumen,
but these results were not found in the Dahl R group.
Cytoplasmic processes are thought to be involved with
transportation of some granules. We believe that exocytosis and the cytoplasmic process would restore the
draining of tissue fluid. The mechanism of these changes
in the structure of lymphatics in the hypertensive heart
needs further investigation.
Cardiac Lymphedema and Myocardial Fibrosis
Recent research has shown that the dominating
motive force of cardiac lymph drainage originates from
not only the external systole and diastole of heart but
also the internal contraction of smooth muscle of the collecting lymphatics. We demonstrated that expression of
ANP was increased in the atrium in the Dahl S group
compared with the Dahl R group. With regard to the
effect of ANP on the lymphatics, some studies have
reported that ANP seems to inhibit lymph transport
through a reduction of spontaneous contractions and a
marked relaxation of lymphatic smooth muscles (Ohhashi et al., 1990; Anderson et al., 1991; Atchison and
Johnston, 1996). We observed extensive dilatation of the
collecting lymphatics in the subepicardium in Dahl S
rats, which may be due to an effect of ANP on the
smooth muscle of the collecting lymphatics. The dilatation of the collecting lymphatics leads to an inability of
the valves to prevent retrograde lymph flow (Ji, 2005),
and consequently, enhanced lymphedema. Connexin43 is
a type of gap junction protein, which is found in the
intercalated disk of the myocardium. It is associated
with the transfer of impulses between adjacent cardiac
muscles (Lin et al., 2006; Kostin, 2007). In our study, we
found a decrease in expression of connexin43 in the
Dahl S group when compared with the Dahl R group,
which indicated that impulse transfer was impeded.
Transmission electron microscopy of the cardiac muscle
also showed serious damage such as the dissolution and
1618
LI ET AL.
rupture of myofibrils, as well as swelling and vacuole
changes in the mitochondrion. These findings demonstrated obstruction of both the transmission of impulses
and the contraction ability of myofibrils.
The findings in this study indicate the obstruction of
lymph drainage in the hypertensive heart. Increased collagen fibers around the lymphatics also limited the opening of intercellular junctions. The drainage of lymph to
the vein as well as tissue fluid to the lymphatics were
all inhibited, which eventually led to lymphedema. A series of structural changes occurred in cardiac lymphatic
endothelium to restore the drainage of lymph and tissue
fluid, such as an increase in vesicles and occurrence of
vacuoles, and active exocytosis and cytoplasmic processes. These changes maintained the balance between
input and output of tissue fluid in the myocardium. If
this balance is disrupted, myocardial fibrosis will become
more severe.
In conclusion, our study demonstrates widespread cardiac fibrosis in the hypertensive heart of the Dahl S
group. This resulted in changes in the structure and
function of the cardiac lymphatics, which should restore
tissue fluid drainage that was impeded by myocardial fibrosis. The cardiac lymphatics were involved in myocardial fibrosis and were affected by myocardial fibrosis.
The lymphatics may be a promising target for treatment
of fibrosis in the hypertensive heart.
ACKNOWLEDGMENTS
The authors thank Ji Ruicheng, Yasuda and Kawazato
of Oita university for their excellent assistance.
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