Recent Advances in the Research of Lymphatic Stomata

Authors


Abstract

Lymphatic stomata are small openings of lymphatic capillaries on the free surface of the mesothelium. The peritoneal cavity, pleural cavity, and pericardial cavity are connected with lymphatic system via these small openings, which have the function of active absorption. The ultrastructure of the lymphatic stomata and their absorption from the body cavities are important clinically, such as ascites elimination, neoplasm metastasis, and inflammatory reaction. The lymphatic stomata play an important role in the physiological and pathological conditions. Our previous study indicated for the first time that nitric oxide (NO) could regulate the opening and absorption of the lymphatic stomata. It could decrease the level of free intracellular calcium [Ca2+] through increasing the cyclic guanosine monophosphate (cGMP) level in the rat peritoneal mesothelial cells, thus regulating the lymphatic stomata. This process is related with the NO-cGMP-[Ca2+] signal pathway. In this review, we summarize the recent advances in understanding the development and the function of the lymphatic stomata. The ultrastructure and regulations of the lymphatic stomata are also discussed in this review. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.

Von Recklinghausen (1863) initially discovered stomata-like structure on the surface of mouse peritoneum by using silver nitrate staining. Later, Baradi et al. (1976) found crater-like stomata in the diaphragmatic pleura and serous membrane of abdomen in mice. The lymphatic stomata were also found in human diaphragmatic peritoneum (Li and Yu, 1990, 1991), human falciform ligament of liver (Tesch et al., 1990), human pelvic peritoneum (Li et al., 1997), fetus meninges (Li and Chen, 1998), frog cardiac pericardium (Li et al., 2000a), the surface of human ovaries (Sui and Li, 2001a), and rabbit costal pleura (Wang, 1975; Li and Li, 2003). The lymphatic stomata have the function of absorption and immunoregulation, and they are associated with various physiopathological processes such as the absorption of ascites (Li, 1992), dissemination of infectious microbes in peritoneal cavity (Leak and Rahil, 1978), and metastasis of tumor cells (Fuantsu et al., 1998).

ULTRASTRUCTURE OF LYMPHATIC STOMATA

Observation under Scanning Electron Microscope

Under scanning electron microscope (SEM) the serous membranes are covered with flat and cubic mesothelial cells. The appearance of both cell types is different. Flat mesothelial cells have larger volume with long and conferted microvilli, which result in the inaccessibility to the cellular surface and tight connection between cells without lymphatic stomata. On the contrary, the cubic mesothelial cells have smaller volume, with “paving stone-like” appearance. The microvilli on the surface of mesothelial cell are short and rarefactive. The protruding digital cytoplasmic processes from the cell body connect with the processes from the consecutive cells to form round or oval lymphatic stomata (Fig. 1). Sometimes filament-like cytoplasmic bridges and microvilli from mesothelial cells can stretch over the lymphatic stomata (Fig. 2). On the mesothelial surface of the serous membrane, there are milky spots made up of macrophages and few lymphocytes. The lymphatic stomata are distributed nearby these milky spots (Fig. 3). The lamellar fibrous connective tissue can be exposed after mesothelial cells are ablate by using sodium hydroxide (NaOH) digestion method (Shimada et al., 1995; Li and Li, 2003). In the lymphatic stomata area, there are many macula cribriformis in the connective tissue under mesothelium. The macula cribriformis is distributed irregularly, and each macula cribriformis consists of many round or oval foramina. The number of foramina is more than that of lymphatic stomata with the diameters ranging 3–5 μm in mice (Shimada et al., 1995). Furthermore, through the foramina of the macula cribriformis, the cytoplasmic processes of lymphatic endothelial cells can be seen. The macula cribriformis is capable of filtering some absorbed substances and considered to be an essential structure responsible for the absorption of fluids and cells (Fig. 4).

Figure 1.

Scanning electron microscopic view to show the two kinds of mesothelial cells: flat mesothelial cells (FM) and cubic mesothelial cells (CM). The round and oval lymphatic stomata are indicated by arrows.

Figure 2.

Scanning electron microscopic view to show the lymphatic stomata (arrow) located at the junction of the four mesothelial cells (M). An endothelial cell process crosses the space below the lymphatic stomata.

Figure 3.

Scanning electron microscopic view showing the milky spots (MS).

Figure 4.

Scanning electron microscopic view to show the exposed connective tissue under the mesothelium (M), after being ablated by using sodium hydroxide. In the distribution area of lymphatic stomata, there are round or oval foramina (arrowheads).

Observation under Transmission Electron Microscope

Under transmission electron microscope (TEM), the lymphatic stomata are known to exist between the cubic mesothelial cells, while connective tissue under mesothelial cells is lacking (Li and Chen, 1992). Through the observation of ultrastructure of flat and cubic mesothelial cells, it was found that there were many differences between them. The nuclei in flat mesothelial cells are round or oval, the organelles are located in the center of cells nearby the nuclei. In flat mesothelial cells, there are few mitochondria and rough endoplasmic reticulum with underdeveloped golgi bodies and rare microfilaments and microtubules. On the contrary, the nuclei in cubic mesothelial cells are bigger, with apparent nucleoli and plentiful mitochondria, rough endoplasmic reticulum, developed golgi bodies, and microtubules and sarciniform microfilaments. This indicates that cubic mesothelial cells have active metabolism. Inside the lymphatic stomata, some valve-like prominences are stretched out from cubic mesothelial cells with pinocytosis alveolus open in the lymphatic stomata. The cytoplasmic processes from the lymphatic endothelial cells extend into tubules under peritoneum, resulting in the complete close of lymphatic stomata and forming closed lymphatic stomata. There are lymphatic lacunas in the deeper lymphatic stomata, that is, cecum of lymphatic vessels. The connective tissue tunnels between the bottom of lymphatic stomata and the top of lymphatic lacunas are called subserous channels. Serous cavities communicate directly with lymphatic system via lymphatic stomata (Fig. 5).

Figure 5.

Transmission electron microscopic view. The pleural cavity (P) is connected with lymphatic capillary (LC) through the stomata (S), subpleura, and the gap between the endothelial cells (E) indicated by arrows. The connective tissue (CT) and mesothelial cells (M) are also shown. (Li and Li, Microsc Res Tech, 2003, 62, 240–246).

THE GENESIS AND DEVELOPMENT OF LYMPHATIC STOMATA

Mesothelial cells originate from fetal mesoderm and begin to differentiate 8–19 days after fertilization (Steven and Mutsaer, 2004). Experiments in our laboratory first showed that human diaphragm lymphatic stomata could be observed in the cubic mesothelial cells as early as in week 20–32 of pregnancy (Li and Yu, 1991). We studied the genesis and development of lymphatic stomata of diaphragmatic peritoneum at NIH strain mice in embryonic period and various postnatal stages using TEM and SEM as well as enzymohistochemistry (Li and Li, 2003). The results showed that on embryonic day (E) 13, the peritoneum only consists of flat mesothelial cells. On E15, some cubic mesothelial cells and early peritoneal lymphatic stomata appeared among the flat mesothelial cells. On E18, diaphragm lymphatic vessels appeared, but trypan blue absorption test failed to show the absorptive function of early peritoneal lymphatic stomata. On the postnatal day (P) 1 subperitoneal lymphatic vessels communicated with lymphatic stomata, and, therefore, the turnover pathway of substance in the peritoneal cavity was established. The postnatal peritoneal lymphatic stomata possess absorptive function, as showed by trypan blue absorption test. However, it has also been reported that lymphatic stomata were first noted on P0 on the peritoneal surface of the diaphragm at ddY strain mice (Toshio et al., 1996). Toshio et al. (1997) elucidated the morphological development process of lymphatic stomata on diaphragm through the observation of peritoneum on E18 and on P0, 4, and 10 under TEM. On E18, the peritoneal surface of diaphragm was covered with a monolayer of mesothelial cells, which attached onto basal membrane by tight or adhesion junction between cells. The cells were flat, with few of microvilli and rarefactive basal membrane under which lymphatic vessels had appeared. On P0, the cytoplasmic prominence extended a little into the connective tissues of basal membrane. On P4, it was apparent that the cytoplasmic prominence of lymphatic endothelial cells deeply extended into the connective tissues, and the tight junction between endothelial cells is still visible. On P10, the cytoplasmic prominence of lymphatic endothelial cells penetrated the connective tissues of basal membrane and got in touch with mesothelial cells, promoting the recovery of flat mesothelial cells and their transformation into cubic mesothelial cells. When the edges of mesothelial cells cohered onto the ends of cellular prominence of lymphatic vessels, lymphatic stomata formed (Fig. 6). The studies on the genesis and development of the lymphatic stomata are of theoretical importance for their functional research and clinical application. However, current studies are still only limited to the morphological observations of the lymphatic stomata with limited data regarding their molecular mechanisms for the regulation and development.

Figure 6.

An ideograph of the development of lymphatic stomata (LS). Stage 1: Appearance of lymphatic vessel (L). Stage 2: The neighboring lymphatic endothelial cells (E) protruded cytoplasmic processes into the submesothelial connective tissue. Stage 3: The lymphatic stomata are formed by the disassembly of the intercellular junctions. P, peritoneal cavity; M, mesothelial cells.

FUNCTION OF LYMPHATIC STOMATA

The lymphatic stomata play an important role in human body in their physiological and pathological conditions. In the physiological conditions, the lymphatic stomata serve as the main drainage channels for the absorption from the serous cavities and maintain the homeostasis of serous cavities. Furthermore, the lymphatic stomata are the direct channel for macrophages migrating from the lymphatic vessel system into serous cavities. Under the pathological conditions, the lymphatic stomata provide the escape route for the ascites, tumor cells, and infections from the serous cavities. Clinically, the lymphatic stomata provide the bases for the absorption of ascites, intraperitoneal chemotherapy, and for the transfusion of blood cells for the therapy of rhesus (Rh) hemolytic fetus.

Active Absorptive Function

Abundant reports have showed that the fluid from the serous cavities is mostly drained through the lymphatic absorption, with only minor part diffused into the blood vessels (Miserocchi et al., 1982; Gotloib and Shustack, 1987). The lymphatic stomata provide rapid direct drainage from the serous cavities into the subjacent lymphatic lacunae. The findings by Leak et al. (1978) showed that bacteria and pharmaceutical particles could be absorbed rapidly via lymphatic stomata. There are many reports that India ink injected into the peritoneal cavity of living animals could be absorbed into the lymphatic capillaries so that the subperitoneal lymphatic vessels were stained dark (French et al., 1960; Tsilibary and Wissig, 1987; Oya et al., 1993). Experiments from our laboratory showed that trypan blue and mouse red blood cells were absorbed by subperitoneal lymphatic vessels via lymphatic stomata, after being injected intraperitoneally (Li and Yu, 1991). It was also reported that cancer cells were able to metastasize via lymphatic stomata (Namba, 1989; Funatsu et al., 1998). Taken together, the lymphatic stomata have active absorptive function. Meanwhile, the one-way flow of absorbed substance from body cavity to lymphatic vessels is maintained by the valve prominence extended from mesothelial cells and the cytoplasmic processes from lymphatic endothelial cells. In general, all substances such as fluid, particles, infective microbes, and tumor cells can be absorbed by lymphatic system via lymphatic stomata all over the body.

Immune Function

Milky spots mainly consist of macrophage and lymphocyte aggregation, while peritoneal milky spots are the first immune barrier in peritoneal cavity, which acts as a source of free macrophages (Shimotsuma et al., 1990, 1991). Abundant milky spots are distributed around lymphatic stomata. The macrophages consisted of milky spots may migrate from lymphatic vessel system via peritoneal lymphatic stomata to peritoneal cavity, or vice versa (Mironov et al., 1979; Cranshaw and Leak, 1990). The milky spots around peritoneal lymphatic stomata may possibly be formed by the resting macrophages, which migrate through amoeboid movement out of lymphatic stomata via subperitoneal lymphatic vessel system. Milky spots play important immune functions in peritoneal cavity (Li et al., 1996a). Shimotsuma et al. (1991) found that when milky spots of human greater omentum were stained with active carbon suspension in vivo, a lot of carbon particles were phagocytized by milky spot cells, indicating that milky spots possess strong nonspecific immune function. In addition, they injected mice peritoneal cavity with inactivated streptococci OK-432 and found that 4 hr postinjection the volume of macrophages increased, together with appearance of rough reductus on the cellular surface. Meanwhile, transmigration of macrophages from lymphatic vessels via the peritoneal lymphatic stomata to peritoneal cavity was observed (Shimotsuma et al., 1992). Increase in the number of macrophages, alteration in their appearance, and polar contribution all indicate that the activation of peritoneal macrophages plays an important role in antitumor (Fujita, 1989). Currently, it is well accepted that macrophages are able to transmigrate between lymphatic vessels via the lymphatic stomata and peritoneal cavity, thereby playing an important role in localizing the inflammation in the peritoneal cavity and preventing the tumor cells metastasis.

THE CONTROLLABILITY OF LYMPHATIC STOMATA

It is common that some cytoplasmic processes derived from mesothelial cells of peritoneal lymphatic stomata, lymphatic endothelial cells as well as collagenoblasts of connective tissue extend into subperitoneal vessels and form mobile valve-form structure, which regulate the absorption of peritoneal lymphatic stomata and maintain the one-way flow of the absorbed substance from peritoneal cavity to lymphatic vessels. Via the peritoneal lymphatic stomata, peritoneal cavity communicates directly with lymphatic system. In the cytoplasm of mesothelial cells, which consist of peritoneal lymphatic stomata, there are a lot of fascicular microfilaments of actin with about 5 nm of diameter. Tsilibary and Wissig (1983) treated mesothelial cells with cytochalasin D to depolymerize intracellular microfilaments and selectively break microfilaments of actin, resulting in apparent alteration of cellular morphology. Later, when cytochalasin D was washed off with normal saline, the morphology of mesothelial cells restored to normal. They thought the microfilaments were essential for the maintenance of normal morphology of lymphatic stomata and constituted the structural basis of the controllability of lymphatic stomata.

The study by Tsilibary and Wissig (1983) demonstrated that whether the lymphatic stomata of diaphragmatic peritoneum was open or close depended on respiratory movement. During inspiration, the diaphragmatic muscles contract, the number of open lymphatic stomata was decreased. On the contrary, during expiration, the diaphragmatic muscles relaxed, the number of open lymphatic stomata was increased. The intraabdominal pressure affects the opening of lymphatic stomata as well (Li et al., 1996b). The increase of intraabdominal pressure leads to increased number of open lymphatic stomata. The study by Li et al. (2001) showed that vascular endothelial growth factor (VEGF) and angiotensin II (Ang II) increased the number of open lymphatic stomata in frog pericardia with enlarged diameter and denser distribution, indicating their efficient regulatory effects on lymphatic stomata in pericardia. Our studies showed that pregnancy, intervention with ovarian stimulation hormones or androgenic hormones were able to affect the opening and absorption functions of lymphatic stomata of ovarian bursa in guinea pig (Sui and Li, 2003). Pregnancy promoted the opening of lymphatic stomata of ovarian bursa, leading to increased absorption function. On the other hand, compared to the pregnancy group and proovulation group, the opening and absorption function of lymphatic stomata of ovarian bursa in androgenic hormone group were decreased.

NO plays an important role in the regulation of lymphatic stomata. Our studies showed that NO generated from macrophages induced by peritoneal dialysates had the effects of relaxing lymphatic vessels, enlarging lymphatic stomata, leading to the enhanced absorption of ascites (Li et al., 2000b). It was also demonstrated that Chinese herbal medicine Atractylodes macrocephala and Salvia miltiorrhiza, which were used to treat ascites in hepatic cirrhosis, enhanced the absorption of ascites through increasing the concentration of endogenous NO to promote the opening of lymphatic stomata (Li et al., 2002a). Doboszynska et al. (2001) demonstrated that the cubic mesothelial cells in the concentrated lymphatic stomata area in broad ligament of uterus of pig had the activity of nitric oxide synthase (NOS) and presumed that NO had the effects on the physiological regulation of lymphatic stomata. In our studies (Li and Li, 2003, 2004, 2008) on the ultrastructure of pleural lymphatic stomata and the lymph draining channels and the mechanisms of signal transduction of lymphatic stomata regulated by NO, it was showed that NO had effects on the cGMP and intracellular free [Ca2+] mesothelial cells, that is, NO could decrease the level of intracellular free [Ca2+] mesothelial cells through increasing the level of cGMP. For the first time, we reported that NO could regulate the opening and absorption of lymphatic stomata via the pathway of NO-cGMP-[Ca2+], providing the important experimental data for the clinical therapy involving drug-regulated opening of lymphatic stomata. This promotes the absorption of ascites in hepatic cirrhosis and also improves the therapeutic effects of peritoneal dialysis. Research on the regulation of lymphatic stomata is the most active topic in the field. Advanced studies on this will be of important theoretical significance and clinical application involving such aspects as absorption of hydrothorax and ascites, the mechanisms underlying the treatment of ascites with traditional Chinese medicine, development of new drugs, as well as the therapies of peritoneal inflammation and cancer.

THE DISTRIBUTION AND CLINICAL SIGNIFICANCE OF LYMPHATIC STOMATA

Lymphatic stomata are small openings of lymphatic capillary on the free surface of the mesothelium, which are widely distributed on different serous membranes in mammals. However, the distribution of lymphatic stomata in the serous membranes is not uniform. There is still some disagreement regarding the distribution of lymphatic stomata on the serous membranes in different species and even in same species.

Peritoneal Lymphatic Stomata

Peritoneal lymphatic stomata are the openings of lymphatic vessels in peritoneal mesothelium by which the peritoneal cavity links up with subperitoneal lymphatic vessels system. The peritoneal lymphatic stomata distribute extensively, mainly on the diaphragmatic surface (Tsilibary and Wissig, 1977; Li and Yu, 1990, 1991; Fukuo et al., 1990; Negrini et al., 1991), falciform ligament of liver (Tesch et al., 1990), visceral peritoneum of ovaries surface (Sui and Li, 2001a), and peritoneum of pelvic wall (Li et al., 1997). Peritoneal lymphatic stomata are round or oval, with few cases being irregular and all of them are in maldistribution and cluster or bendiformis, which are located among the cubic mesothelial cells.

Peritonitis, hepatic cirrhosis, and peritoneal cancer can all induce ascites. A great quantity of ascites may press important organs as heart and lung, often leading to the consequences endangering patient's life. Clinically, the traditional therapies for drawing the ascites include catharsis, diuresis, and others. All these have no significant curative effects and have considerable side effects and may also lead to an opportunistic infection. In contrast, peritoneal lymphatic stomata have active absorption function so that fluid, particles, cells, and even infective microbes and tumor cells may all be absorbed rapidly via peritoneal lymphatic stomata (Li and Li, 2000). Tsilibary and Wissig (1987) proposed a concept of lymphatic drainage units, which are composed of a lymphatic lacuna, a covering of lacunar mesothelium, and intervening submesothelial connective tissue. The authors pointed out that during the turnover of ascites, lymphatic drainage units played an important role. Further studies showed that when ascites exists, with the increase of intraabdominal pressure, the number of open lymphatic stomata increased with larger aperture. Through the screening of a great quantity of traditional Chinese medical herbs (Salvia miltiorrhiza, Codonopsis pilosula, Alisma rhizome, Atractylodes macrocephala, etc.), which were found to have therapeutic effects on ascites, we demonstrated that these herbs were able to promote the opening of peritoneal lymphatic stomata, leading to enhanced absorption of ascites via peritoneal lymphatic stomata (Li et al., 1996b).

Continuous ambulatory peritoneal dialysis (CAPD) has become a common substitution therapy of chronic renal insufficiency, but during long-term CAPD, pure ultrafiltration will decrease and even cause the failure of the peritoneal dialysis due to the persistent reabsorption of the peritoneal lymphatic stomata (Li and Yu, 1994). To prevent this dysultrafiltration, it is beneficial to add the drugs to the dialysate so as to contract the peritoneal lymphatic stomata and, therefore, reduce the reabsorption.

Currently, the peritoneal lymphatic stomata are used successfully in the therapy of Rh hemolytic fetus (Li and Yu, 1994). New trails are being made in maintaining the peritoneal homeostasis and developing new herbal drugs for the treatment of ascites, peritonitis, and peritoneal cancer.

Pleural Lymphatic Stomata

Pleural lymphatic stomata distribute mainly on the pleural membrane between ribs whose front side gets close to chest bone, while rear side close to backbone and the flank of bony thorax, with the tendency that the density of distribution increases from top to bottom. In the pleural mesothelium of the murine diaphragm, there are very few cuboidal mesothelial cell areas and lymphatic stomata (Fukuo et al., 1990; Ohtani et al., 1993). However, there are numerous lymphatic stomata in both the peritoneal and pleural surfaces of the rabbit diaphragm (Negrini et al., 1991). There are no lymphatic stomata on the pulmonary pleura, mediastinal pleura, and the pleura covering the costal bone. Therefore, only the flat mesothelial cells are distributed on the costal pleura, and there are no lymphatic stomata as well. There are round or oval lymphatic stomata on the pleura between the costal bones scattered or clustered in distribution (Li and Li, 2004). Silky cytoplasmic bridges and microvilli from mesothelial cells stretch across lymphatic stomata; meanwhile, valve-like cytoplasmic processes from lymphatic endothelial cells extend into the vessels. In some situations, lymphatic stomata are sealed completely by valve-like cytoplasmic processes, resulting in closed lymphatic stomata. From pleural lymphatic stomata connective tissue fibers on the bottom can be seen. Pleural cavity links up with lymphatic lacuna cavities through subpleural ductules and interstitial spaces of endothelial cells inside lymphatic vessels via lymphatic stomata, forming the communication among lymphatic system of pleural cavity and constituting important route directly linking pleural cavity with vessel system.

Pleural lymphatic stomata are important routes for the draining of pleural cavity lymph that play a crucial role in the formation and turnover of hydrothorax and the pleural metastasis of tumor and infective microbes. To determine the contribution of the parietal lymphatic vessel to pleural liquid and protein removal through stomata, Broaddus et al. (1988) instilled autologous protein solution with labeled albumin and erythrocytes through the capsule into the pleural space of sheep with very large hydrothorax, which can only leave through the stomata owing to their size. They got the conclusion that the lymphatic drainage through the stomata contributed 89% of protein and liquid removal from the pleural space. Further study has confirmed that lymphatic drainage through the stomata did not contribute most of protein and liquid removal from the pleural space under physiological condition, but with the high hydrothorax it contributed 64% of the removal of labeled albumin from the pleural space (Bodega and Agostoni, 2004). There is other study showing that transcytosis might contribute a relevant part of protein and liquid from the pleural space (Agostoni et al., 2002). Study on pleural lymphatic stomata is of importance for the clinical application. For example, mechanisms underlying pleural effusion may involve: the amount of pleural effusion exceeds the capability of draining via pleural lymphatic stomata; lymphatic stomata are blocked by cell debris, proteins, or fibers during the conditions such as cancer or other malignant diseases in thoracic cavity. The lymphatic stomata can be obstructed by thick fibrous layers that appear in lupus erythematosus, rheumatoid arthritis, and other diseases. Thoracic tuberculosis, metastatic tumors, infection, and adenomyosis are more frequently seen in the right thoracic cavity, which probably could be attributed to more quantity of lymphatic stomata in the right (Li et al., 1996b). On the other hand, thoracic and abdominal cavities can communicated each other by diaphragm lymphatic stomata. Ohtani et al. (1997) reported that celiac cancer cells were found in drained hydrothorax in rat. Study on pleural lymphatic stomata is of clinical importance for exploring the mechanisms of lymph draining in pleural cavity, the formation and turnover of hydrothorax, and the pleural metastasis of tumor and infective microbes.

Pericardial Lymphatic Stomata

Cardiac pericardium consists of two layers of mesothelial cells together with a lamellar connective tissue between them. The side facing pericardial cavity is called pericardial side, while that facing thoracic called thoracic side. Pericardial lymphatic stomata are found only in pericardial side and are scattered in the round or oval shapes in most cases (Li et al., 2000a). Pericardial lymphatic stomata link up with submesothelial lymphatic vessels of cardiac pericardium, therefore communicating pericardial cavity with vessel system and forming the direct route of pericardial fluid from pericardial cavity into vessel system. In clinical setting, pericardium can be involved by inflammation, trauma, and tumor. Also, various diseases such as tuberculosis and viral infection can cause stagnation of cardiac venous and lymphatic systems, resulting in pericardial effusion owing to leakage of cardiac interstitial fluid via epicardium and retention in pericardial cavity (Stewart et al., 1997). In physiological status, pericardial lymphatic stomata open alternately to run the physiological circulation of pericardial fluid in pericardial cavity (Li et al., 2002b). We injected VEGF and Ang II into the peritoneal cavity of frog and then observed the cardiac pericardia under SEM. It turned out that VEGF and Ang II increased the number of open pericardial lymphatic stomata and enlarged the pore size, indicating that they had significant regulatory effects on pericardial lymphatic stomata (Li et al., 2001). In clinical, there is no effective therapy for pericardial effusion. As it is known that pericardial lymphatic stomata are involved in microcirculation of cardiac lymph system and play an important role in lymph draining, advance study on the mechanisms regarding pericardial lymphatic stomata on the turnover of pericardial effusion will be helpful for the clinical therapy of pericardial effusion.

Ovarian Bursa Lymphatic Stomata

For the first time we reported the existence of human ovarian lymphatic stomata (Sui and Li, 2001a) and guinea pig ovarian bursa lymphatic stomata (Sui and Li, 2001b). Ovarian lymphatic stomata are associated with ovary lymphatic vessels and the transportation of substance in peritoneal cavity, and, further, it has the function of local immunity and may affect reproduction function. Mesovarium is the peritoneum that links ovary and broad ligament and its development varies considerably. Most rodents have developed mesovarium and form a capsular structure enveloping ovary called ovarian bursa. In contrast, primate and human being hardly have mesovarium so that their ovaries are exposed in peritoneal cavity extensively (Zhang and Li, 2005). Murine ovarian bursa consists of interior and exterior layers of mesothelial cells together with the central connective tissue, with the thickness of about 30 μm. The interior epithelia of ovary bursa consist mainly of overlapping flat and cubic epithelia, and the lymphatic stomata of ovarian bursa are located among cubic epithelia and occasionally among flat epithelia, with the clustered or scattered distribution. The lymphatic stomata are derived from cytoplasmic processes of nearby cells with smooth and clear edges in various sizes and in round, oval, or irregular shapes. Lymphatic stomata link up with lymph lacuna, constituting direct routes communicating among ovary bursa, lymphatic system, and peritoneal cavity (Zhang and Li, 2005). The surface of human ovary is uneven, covered with both flat and cubic mesothelial cells. The ovarian lymphatic stomata exist in the cubic mesothelial cells, with the clustered or scattered distribution. Meanwhile, the distribution of ovarian lymphatic stomata can be observed in the symphysis between flat and cubic mesothelial cells, consisting of cytoplasmic processes from the two kinds of cells.

In rodents ovarian bursa play an important role in ovulation and breeding of the animals, and the key structure executing the functions is the lymphatic stomata of ovarian bursa (Li et al., 2007). The ovarian lymphatic stomata possess the functions of active absorption and key draining, providing crucial ways for transportation of substance via in tela lymph system and serous membrane, which are of importance for the maintenance of humor homeostasis around ovary and may affect the reproductive function of ovary. Ovarian bursa lymphatic stomata possess immune function as well. Macrophages and lymphocytes can transmigrate via lymphatic stomata between lymph system and peritoneal cavity and both cells can form milky spots structures, which play an important role in immune functions. Ovarian lymphatic stomata are of important significance for ovary lymph microcirculation and maintaining ovary microenvironment homeostasis and promoting the growth of ovarian follicles and the development of luteum. Further studies on the biology of ovarian bursa lymphatic stomata will possess the important clinical significance involving peritoneal metastasis of ovarian tumor, ascites formation derived from ovarian tumor and its turnover, as well as exairesis of ovarian tumor.

Meninges Lymphatic Stomata

Through the study on fetus meninges, it was found that there were lymphatic stomata on human meningina and scleromeninx (Li and Chen, 1998). Meninges lymphatic stomata are located in between meninges mesothelial cells, in round, or oval shapes, with the clustered or scattered distribution. It was showed that the density of meninges lymphatic stomata on scleromeninx was greater than that on meningina. It is still not demonstrated that there are lymphatic vessels in central nervous system, but lymph draining indeed exists. Cerebral lymph draining is of importance for the maintenance of normal cerebellar physiological functions and preventing “lymphostatic encephalopathy” (Foldi, 1977). Intracranial lymph and tissue fluid can communicate with cervical lymphatic vessels via cerebellar prelymphatic vessel system, executing cerebellar lymph draining. “Prelymphatic vessels” refer to intracal cellular route, which has functions similar to lymphatic vessels but morphologically has not endotheliocytes on the vessel wall. The existing study showed that the Indian ink injected into subarachnoid cavity could be found in nose lymphatic vessel and cervical glands, but there was no morphological evidence (Zhang et al., 1992). The discovery of meninges lymphatic stomata provided new data for the study of cerebellar lymph turnover. Meninges lymphatic stomata may be a part of cerebellar prelymphatic vessel system, which have important effects on cerebrospinal fluid circulation and are involved in cerebellar normal physiological function.

Tumor Metastasis

Namba et al. (1989) injected VX2 cancer cells into the peritoneal cavity of rabbit and then under electron microscope they found decreased cellular cytoplasm, shrinkage of cells, breakage of cell junction, and enlarged pore size of lymphatic stomata. Furthermore, it was found that cancer cells got into lymphatic vessels via lymphatic stomata. Our study showed that there existed lymphatic stomata on the capsule walls of human ovarian mucinous cystadenoma, which provided the morphological evidence for the early metastasis of the ovarian tumor cells into the peritoneal cavity via ovarian lymphatic stomata (Sui and Li, 2001a). It is of important significance that tumor cells metastasize and disperse via lymphatic stomata. Metastasis of tumor cells via lymphatic stomata is one of the determinant factors threatening survival of patients. Therefore, studies on the mechanisms underlying the metastasis of tumor via lymphatic stomata will be helpful for the targeted therapy of cancer and effective inhibition of tumor metastasis. Establishment of the coculture system in vitro for tumor cells, mesothelial cells, and lymphatic endothelial cells will provide an important experimental model for the studies on molecular regulation mechanisms of lymphatic stomata, metastasis of tumor via lymphatic stomata, development of new antitumor drugs, and clinical therapy of metastatic tumor.

PROSPECT

The lymphatic stomata have important functions in the absorption of ascites, cancer metastasis, and the regulation of the peritoneal homeostasis. However, there are still many questions to be answered, such as the molecular mechanisms of the lymphatic stomata development, intracellular signal transduction, cancer metastasis via the lymphatic stomata, as well as the regulatory effects of drugs and cytokines on the lymphatic stomata. Further studies on the molecular mechanisms regarding the lymphatic stomata development, transformation of the flat mesothelial cells to the cubic mesothelial cell will provide evident theoretical basis for the clinical application of the lymphatic stomata in the new drug development for the pleural, pericardial, and peritoneal effusion, for prevention of the cancer metastasis, and the dissemination of infective microbes in the body cavity.

Ancillary