Biological Characteristics of Lymphatic Endothelial Cells with Special Reference to Nitric Oxide and Lymphangiogenesis

Biological Characteristics of Lymphatic Endothelial Cells with Special

Reference to Nitric Oxide and Lymphangiogenesis

Toshio Ohhashi

Summary.

Biological properties of lymphatic endothelial cells including measurement of partial pressure of oxygen, establishment of rat lymphatic endothelial cell line, immunoreactivity of endothelial constitutive nitric oxide (NO) synthase in the cultured lymphatic endothelial cells, NO-mediated mod- ulation of spontaneous contractions in isolated lymph vessels and lymph pump activity in vivo, and flow-mediated release of NO from lymphatic endothelial cells were shown with our current studies. By using the cultured lymphatic endothelial cells, basic fibroblast growth factor-mediated lym- phangiogenesis in vitro is also demonstrated.

Key words.

Po

in lymph, Lymphatic endothelial cell line, Nitric oxide, Lymphangiogenesis, Basic fibroblast growth factor

Introduction

The lymphatic system returns fluid and protein to the circulation by mecha- nisms that are not completely understood. How are fluid and protein trans- ported from the tissue spaces into the lymph capillaries (lymph formation)?

Many new theories are being developed but the problem is complicated by our uncertainty about tissue fluid pressure. Once lymph is formed, how is it transported back to the general circulation? Some lymph vessels are known to contract intrinsically, and the contraction may play a significant role in the centripetal propulsion of lymph [1–3]. We have studied the physiology and pathophysiology (lymph edema and lymphatic metastasis of carcinoma cells) of lymph formation and lymphatic transport. This chapter summarizes our

267 Department of Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan

findings in four areas: (1) the partial oxygen pressure of lymph in vivo, (2) establishment of rat lymphatic endothelial cell line, (3) functional roles of nitric oxide (NO) in lymphatic transport, and (4) basic fibroblast growth factor (bFGF)-mediated lymphangiogenesis in vitro.

Partial Pressure of Oxygen in Lymph In Vivo

There exists a discrepancy regarding on partial pressure of oxygen (Po

) of lymph; some studies showed the values lower than the Po

in venous blood and the others the values similar to the value in venous blood [4]. Thus we measured changes in the Po

of lymph in thoracic ducts in vivo by using oxygen electrodes and then examined the effects of 3 mM KCl on changes in the Po

of lymph and lymph flow rate. Mongrel dogs were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and artificially ventilated with room air by a respirator. A catheter was inserted into the right femoral vein to ad- ministrate physiological saline solution or the high-potassium solution and another one into the right femoral artery to monitor changes in the systemic arterial pressure. A polyethylene catheter connected with a glass capillary and oxygen electrode was inserted into the intrathoracic duct from the left jugular angle. The partial pressure of oxygen in lymph was continuously measured with an oxygen electrode and its electrical amplifier. The flow rate of lymph was also recorded by using a home-made drop-counter. The Po

values of arte- rial and venous blood were 110.5 ± 8.0 mmHg and 55.2 ± 3.5 mmHg, respec- tively (n = 7). As shown in Fig. 1, the Po

value of lymph in the thoracic duct was around 35 mmHg in vivo.

The cardiac arrest produced by an intravenous administration of 3 mM KCl caused a rapid decrease of the arterial pressure that became around 0 mmHg by 1 min after the cardiac arrest. The cardiac arrest also produced a gradual decrease of the Po

value that arrived at a stable value (10 mmHg) by 15 min after the cardiac arrest. In contrast the lymph flow rate was rapidly and magnificently increased by the cardiac arrest, the slight increase of which was significantly kept during 15 min after the cardiac arrest [5]. These findings suggest that lymphatic system work physiologically under 35–40 mmHg in Po

.

Establishment of Rat Lymphatic Endothelial Cell Line

The cultured lymphatic endothelial cells of dogs and cows have been used to investigate biological and morphological properties of the endothelial cells [6,7]. No report, however, exists regarding the establishment of lymphatic endothelial cell line from small experimental animals such as rats and mice.

268 T. Ohhashi

The establishment may facilitate the promotion of key studies to evaluate cellular and molecular mechanisms of lymphangiogenesis. Thus we have attempted to establish rat lymphatic endothelial cell line and then to investi- gate morphological and immunohistochemical properties of the cultured cells and arrangement of cytoskeleton protein F-actin [8]. The lymphatic endothelial cells of rat thoracic ducts were isolated enzymatically by trypsin digestion and were cultured in endothelium growth medium (EGM)-2 in an atmosphere of low oxygen (5% O

, 5% CO

, and 90% N

) or high oxygen (21%

O

, 5% CO

, and 74% N

). The number of the cells cultured in the low-oxygen atmosphere (48 750 ± 10 594 cells/ml, n = 4) was significantly larger than that obtained in the high-oxygen atmosphere (4333 ± 1377 cells/ml, n = 4).

The cultured cells in the low-oxygen atmosphere showed a monolayer with

uniform cobblestone appearance, suggesting the morphological properties of

endothelial cells (Fig. 2). Factor VIII-related antigen and cell surface carbo-

hydrates were found on the lymphatic cultured cells. The phagocytosis of

1 ,1-diocadecyl1-3,3,3¢,3¢-tetramethylindo-carbocyanine perchlorate-labeled

acetylated low-density lipoprotein also was observed in the cultured cells. The

cytoskeleton protein F-actin was located on the plasma membrane of the

cultured cells as circumferential thin bundles and in the cytoplasm as fila-

mentous bundles. The study indicates that the choice of EGM-2 as a culture

medium and the hypoxic atmosphere (~5%) enabled us to establish rat lymphatic endothelial cell line.

Physiological Roles of Endogenous NO in Lymphatic System

The immunoreaction to antiendothelial constitutive NO synthase (ecNOS) was significantly positive to the cultured lymphatic endothelial cells (LEC) [9]. When we stained 14 samples of the LEC to the anti-ecNOS, the immunore- active signals were intense in the nucleus and cytoplasm (10 out of 14). In

Fig. 2. Representative photographs of a phase-contrast image (A) and of the arrangement of F-actin (B) in cultured rat lymphatic endothelial cells. The bars in A and B are 100 and 50mm, respectively. From ref. [8]

4 of 14 samples, the intense signal of anti-ecNOS was restricted in the nuclei.

The NO released from lymphatic endothelial cells can regulate the rhythm and amplitude of spontaneous contractions in isolated bovine mesenteric lymph vessels [10]. Regular spontaneous contractions at a constant rate of about 3 beats/min were observed in the isolated lymph vessels. Acetylcho- line (ACh) at concentrations between 10

and 10

M caused both negative chronotropic and inotropic effects on the spontaneous contractions. The ACh- mediated negative effects were completely reversed in all lymphatic segments studied when the endothelium was removed mechanically. Addition of 3 ¥ 10

M N

-monomethyl-l-arginine (l-NMMA) tended to increase the rhythm and amplitude of the spontaneous contractions in the control. The ACh- mediated negative chronotropic and inotropic effects in the lymphatic seg- ments with intact endothelium were significantly reduced by the pretreatment with l-NMMA. An additional treatment with 10

M l-arginine in the same segments caused a complete reversal of the ACh-mediated chronotropic and inotropic effects on the spontaneous contractions. The findings suggest that NO liberated from the lymphatic endothelium seems to inhibit pacemaker activity of the spontaneous contractions and to reduce myogenic conduction and/or the mechanical activity of the lymphatic smooth muscles.

We also examined effects of flow (shear stress) on lymphatic endothelial cells by using conventional cascade bioassay preparations [11]. The pressur- ized canine thoracic ducts were intraluminally circulated at flow rate ranging from 0.5 to 2.0 ml/min. A linear relationship between the flow rate and the normalized amount of NO released from the lymphatic endothelial cells was observed during the range of flow rate. Thus the lymphatic endothelial cells are very sensitive to lowest changes of shear stress compared with arterial and venous endothelial cells.

Next we attempted to examine effects of N

-nitro-l-arginine methyl ester (l-NAME) on the pump activity of rat mesenteric lymph vessels in vivo by using a vital video microscope [12]. The exposed surface of rat mesentery was continuously perfused with 37°C bicarbonate-buffered physiological salt solu- tion. Pumping frequency (PF), end-diastolic (EDD) and end-systolic diame- ters (ESD) of the mesenteric lymph microvessels were measured with the vital microscopic system and then the pump flow index (PFI) was calculated. A 15- min perfusion of 30 mM l-NAME over the mesenteries caused a significant increase of the PF and PFI and a significant decrease of the EDD and ESD.

Simultaneous perfusion of 1 mM l-arginine with 30 mM l-NAME produced a

significant reversal of the l-NAME-mediated increase of PF and decrease of

ESD. The findings suggest that endogenous NO, may be released by the

stimulation of flow, have physiologically modulated the pump activity in rat

mesenteric lymph vessels in vivo.

Basic Fibroblast Growth Factor-Mediated Lymphangiogenesis In Vitro

Elevated interstitial fluid pressure in human tumors has been attributed to the increased permeability of tumor vessels, the growth of the vessels in a confined space, and the absence of a well-defined lymphatic system [13]. We examined whether the cultured lymphatic endothelial cells can induce in vitro neovascularization of lymph vessels, similar to angiogenesis of the blood vessels, in response to basic fibroblast growth factor (bFGF) [14]. The effects of bFGF on the proliferation and migration of cultured lymphatic endothe- lial cells were evaluated by changing the number of the subconfluent cells and by wound migration assay, respectively. We also examined effects of bFGF on invasion of the cultured lymphatic endothelial cells into a three-dimensional collagen gel by using a phase-contrast microscope (Fig. 3) and an electron microscope. The bFGF caused significant invasion and tube formation into the three-dimensional collagen gel. The growth factor also facilitated forma- tion of capillary-like-tubes of the cultured cells between two layers of colla-

Fig. 3. A representative microphotograph demonstrating tube formation (lymphangio- genesis) of the cultured lymphatic endothelial cells when the cells were sandwiched between two layers of type I collagen gels in the presence of 10 ng/ml basic fibroblast growth factor. Bar = 100 mm

gen gels. The findings suggest that the cultured lymphatic endothelial cells can form lymphatic capillary-like tubes in response to bFGF.

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