Ultrastructure Changes of Cardiac Lymphatics During Cardiac Fibrosis in Hypertensive Rats.код для вставкиСкачать
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: firstname.lastname@example.org 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 1614 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). 1616 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. 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