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From the First Department of Physiology, Shinshu University School of Medicine and Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Matsumoto, Japan.
Correspondence: Address correspondence and reprint requests to: Toshio Ohhashi, MD, The First Department of Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390–8621, Japan; Fax: 81-263-36-5149; E-mail: ohhashi{at}sch.md.shinshu-u.ac.jp
ABSTRACT
Biological properties of lymph microvessels include characteristics of spontaneous contractions of lymph vessels, nitric oxide (NO)-mediated modulation of active lymph pump activity, flow-induced production and release of NO from lymphatic endothelial cells, and localization of NO synthase in cultured lymphatic endothelial cells. B16-BL6 melanoma cells release factors that inhibit active pump activity in isolated lymph microvessels. We have found that basic fibroblast growth factor (bFGF) induces significant proliferation and migration of canine cultured lymphatic endothelial cells. This growth factor facilitates the formation of capillary-like tubes by the cultured endothelial cells.
Key Words: Carcinoma cells • Flow stimulation • Lymph vessel • Lymphangiogenesis • Lymphatic endothelial cell • Nitric oxide
The lymphatic system returns fluid and protein to the circulation by mechanisms that are not completely understood. How are fluid and protein transported 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 of lymphatic transport as it relates to lymphatic mapping. This paper summarizes our findings in four areas:1 functional roles of nitric oxide (NO) in spontaneous contractions and active lymph transport;2 establishment of a rat lymphatic endothelial cell line;3 melanoma cell-released inhibitory factor(s) of active pump activity in lymph vessels; and4 basic fibroblast growth factor (bFGF)-mediated tube formation by cultured lymphatic endothelial cells.
SPONTANEOUS CONTRACTIONS OF LYMPHATIC SMOOTH MUSCLES
It has been suggested that spontaneous contractions of lymphatic smooth muscles are responsible for lymph transport in mesenteries under physiological conditions.1–3 The frequency of spontaneous contractions is correlated with wall tension of the lymph vessels. Our group reported that the vibratory stimulation produced spontaneous contractions in quiescent preparations isolated from bovine mesenteric lymphatics.3 We also found that the vibratory stimulation accelerated the rhythm of existing spontaneous contractions.4 The results suggest that the spontaneous contractions of lymphatic smooth muscles are regulated not only by the magnitude of stretch but also by the rate and acceleration of stretch-mediated deformation of the lymphatic walls. The rate and volume of lymph flow in each lymphangion (the functional unit of the lymph vessels between the two valves) may regulate active lymph transport generated by the spontaneous contractions.
NO MODULATES SPONTANEOUS CONTRACTION OF LYMPH VESSELS
Lymphatic endothelial cells, as well as arterial and venous endothelial cells, have the potential to produce and release NO or its related compounds.5 The NO released from lymphatic endothelial cells can regulate the rhythm and amplitude of spontaneous contractions in isolated bovine mesenteric lymphatics.6 Regular spontaneous contractions at a constant rate of about 3 beats/min were observed in the isolated bovine mesenteric lymph vessels. Acetylcholine (ACh) at concentrations between 10-7 and 10-6 M caused both negative chronotropic and inotropic effects on the spontaneous contractions. The ACh-induced negative chronotropic and inotropic effects were completely reversed in all lymphatic segments studied when the endothelium was removed mechanically. Addition of 3 x 10-5 M NG-monomethyl-L-arginine (L-NMMA) tended to increase the rhythm of the spontaneous contractions in the control. The ACh-induced negative chronotropic and inotropic effects in the lymphatic segments with intact endothelium were significantly inhibited by pretreatment with L-NMMA. An additional treatment with 10-4 M L-arginine in the same segments completely reversed ACh-induced chronotropic and inotropic effects on spontaneous contractions. Thus, NO liberated from the lymphatic endothelium seems to inhibit pacemaker activity of spontaneous contractions and to reduce myogenic conduction and/or the mechanical activity of lymphatic smooth muscles.
IMMUNOLOCALIZATION OF NO SYNTHASE IN CULTURED CANINE LYMPHATIC ENDOTHELIAL CELLS
Lymphatic endothelial cells [LEC] isolated from dog thoracic ducts were cultured as previously described.7 Briefly, 10 to 15 cm of the thoracic ducts were isolated and placed in cold Hanks buffered-salt solution (HBSS). Each lymph vessel was flushed with cold HBSS and incubated with collagenase solution for 10 minutes at 37°C. The cells were harvested by washing the vessels with Earle’s minimal essential medium (MEM) containing 10% fetal bovine serum (FBS), collected by centrifugation, and resuspended in complete MEM cultured medium supplemented with 20% FBS, penicillin, streptomycin, and amphotericin B. The lymphatic cell suspension was placed into 35-mm culture dishes that were coated with 0.6 mg/mL collagen type-I.
The cells were identified as lymphatic endothelial cells by indirect immunofluorescence staining of factor VIII-related antigen and uptake of 1,1-diocadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate–labeled acetylated low-density lipoprotein (Dil-Ac-LDL). Primary antisera and normal rabbit immunoglobulin G (negative control) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The immunoreactions to anti-ecNOS and iNOS were significantly positive to the cultured LEC. When we stained 14 samples of the LEC to the anti-ecNOS, the immunoreactive signals were intense in the nucleus and cytoplasm (10 of 14); in 4 of 14 samples, the intense signal of anti-ecNOS was restricted in the nuclei. The immunoreactivity of anti-iNOS to the LEC was not so intense but was clearly positive to the cytoplasm of the LEC.8
Regulation of the expression of iNOS in lymphatic endothelial cells requires further study, but until recently the literature contained no report regarding establishment of lymphatic endothelial cell lines from small experimental animals such as rats and mice. The establishment of these cell lines would facilitate evaluation of cellular and molecular mechanisms of lymphangiogenesis in vascular endothelium growth factor receptors (VEGFR). We have successfully established a rat lymphatic endothelial cell line.9 In preliminary experiments, we were not able to culture and maintain rat lymphatic endothelial cells in minimum essential culture medium and normoxic atmosphere (about 21% O2). A new culture medium (EGM-2) and low-oxygen conditions (5% O2, 5% CO2, 90% N2) enabled us to establish a rat lymphatic endothelial cell line. The hypoxic conditions may be compatible with the finding that lymphatic endothelial cells work physiologically in an atmosphere of low oxygen (25 to 40 mm Hg).10
PHYSIOLOGICAL ROLES OF ENDOGENOUS NO IN LYMPHATIC PUMP ACTIVITY IN VIVO
Little information exists regarding potential effects of endogenous NO on the pump activity of mesenteric lymphatics in vivo. Thus, we have attempted to examine effects of N
-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine on the pump activity of rat mesenteric lymphatics in vivo by using a vital video microscope. The mesentery was prepared for intravital video microscopic observation as described previously.11 In brief, a midline incision was made and mesentery adjacent to a segment of small intestine was exteriorized. The intestinal segment was positioned in a domestic-made semicircular channel that surrounded an optical window for observing lymphatic pump activity. The collecting lymph vessels (80 µm) chosen for the study were located approximately 10 mm from the intestinal wall. A plastic plate was placed over the intestinal semicircular channel with Dow Corning silicone grease, in a manner that produced a seal between the intestine and mesentery but did not obstruct blood and lymph flow. The exposed surface of mesentery was continuously perfused with 37° to 38°C bicarbonate-buffered salt solution. The preparation was transferred to a video microscope system, by which the pump activity in rat mesenteric lymphatics was continuously monitored and recorded.
Pumping frequency (PF), endodiastolic diameter (EDD), and endosystolic diameter (ESD) of the mesenteric lymph microvessels were measured with the microscopic system, and then the pump flow index (PFI) was calculated. A 15-minute perfusion of 30 µM 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 µM L-NAME produced a significant reversal of the L-NAME-mediated increase of PF and decrease of ESD. A 15-minute perfusion of 100 mM aminoguanidine caused no significant effects on PF, EDD, and ESD of the mesenteric lymph vessels in vivo. These findings suggest that endogenous NO has physiologically modulated the pump activity in rat mesenteric lymphatics in vivo and that the production and release of NO may be mediated by ecNOS but not by iNOS.12
FLOW-MEDIATED RELEASE OF NO FROM LYMPHATIC ENDOTHELIAL CELLS
Recently we examined effects of flow on lymphatic endothelial cells by using conventional cascade preparations of isolated canine coronary arteries without intact endothelium. The pressurized thoracic ducts were intraluminally perfused at a constant flow rate ranging from 0.5 to 2.0 mL/min. A linear relationship between the flow rate and the normalized amount of relaxing substance(s) released from the lymphatic endothelial cells was observed. Thus, a flow of 2.0 mL/min produced about 39% of sodium nitroprusside (SNP)–induced maximal relaxation in the cascade arterial rings. The 10-5 M ACh-induced and flow-induced relaxations of the cascade arterial rings were completely reversed by mechanical rubbing of lymphatic endothelial cells in the pressurized lymph vessels. Pretreatment of lymphatic endothelial cells with 5 x 10-5 M L-NAME significantly reduced ACh-induced and flow-induced vasodilations of cascade arterial rings. Pretreatment of lymphatic endothelial cells with 10-5 M indomethacin produced no significant effect on ACh-induced and flow-induced vasodilations. These findings suggest that lymphatic endothelial cells of canine thoracic ducts are able to produce and release endogenous NO by stimulation of flow (approximately 2.0 mL/min).13
B16-BL6 MELANOMA CELLS RELEASE FACTORS THAT INHIBIT PUMP ACTIVITY IN ISOLATED LYMPH VESSELS
We investigated whether the supernatant from cultured melanoma cell lines B16-BL6 and K1735 or Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity.
Pretreatment with 3 x 10-5 M L-NAME or 10-6 M glibenclamide and 5 x 10-4 M 5-hydroxydecanoic acid significantly reduced supernatant-mediated inhibitory responses. Simultaneous treatment with 10-3 L-arginine and 3 x 10-5 M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous NO plays an important role in supernatant-mediated inhibitory responses. Chemical dialysis of substances of <1000 molecular weight (MW) completely reversed the supernatant-mediated response. In contrast, pretreatment by heating or digestion with protease had no significant effect on the supernatant-mediated response. These findings suggest that B16-BL6 cells may release <1000-MW nonpeptide substances that result in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K+ channels.14
BASIC FIBROBLAST GROWTH FACTOR-MEDIATED LYMPHANGIOGENESIS
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.15 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). The effects of bFGF on the proliferation and migration of cultured lymphatic endothelial cells isolated from canine thoracic ducts were evaluated by changing the number of the subconfluent cells and by wound migration assay, respectively. The effects of bFGF on invasion and tube formation of the cultured lymphatic endothelial cells into a three-dimensional collagen gel were also investigated with a phase-contrast microscope and an electron microscope. bFGF (10 ng/mL) induced significant proliferation and migration of the cultured lymphatic endothelial cells, as well as significant invasion and tube formation of these cells into a three-dimensional collagen gel. bFGF also facilitated the formation of capillary-like tubes of the cultured cells between two layers of collagen gels. These findings suggest that the cultured lymphatic endothelial cells can form lymphatic capillary-like tubes in response to bFGF.7
FOOTNOTES
Basic fibroblast growth factor (bFGF) induces significant proliferation and migration of canine cultured lymphatic endothelial cells. This growth factor facilitates the formation of capillary-like tubes by the cultured endothelial cells.
Received for publication November 24, 2003. Accepted for publication December 10, 2003.
REFERENCES
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