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Currently, there are many methods to evaluate the effectiveness of manual lymph drainage in the treatment of lymphedema, that is, limb volume measurement, bio-electrical impedance measurement, computer tomography, and ultrasound imaging. However, it is difficult for these methods to accurately address the lymph flow generated by manual lymph drainage. Therefore, we aimed at developing a concise and accurate method to measure the lymph flow through the thoracic duct in human subjects, which is applicable for evaluating the effectiveness of manual lymph drainage. In the present mini-review, we demonstrate the developed method in detail and its scientific evidence for the effectiveness obtained with animal and human clinical experiments. In rat

It is known that nitric oxide (NO) is a gas and synthesized from l-arginine by the NO synthase (NOS) in vascular endothelial cells. The diffused NO activates the guanosine monophosphate, which initiates a series of intracellular events, leading to physiological response such as vasodilation. There are three different types of NOS, namely endothelial constitutive NOS (ecNOS), neuronal NOS (nNOS), and cytokine-inducible NOS (iNOS). The ecNOS and nNOS are expressed constitutively at low levels and can be activated rapidly by an increase in cytoplasmic calcium ions. In contrast, the iNOS is induced when macrophages are activated by cytokine, resulting in the induction of pathophysiological effects. Lymph flow is known to stimulate the release of NO from lymphatic endothelial cells (LEC) and then produce the relaxation of lymphatic smooth muscle cells. The NO also plays a key role in the control of lymphatic pump activity

We demonstrated pulmonary arteriolar blood flow-mediated CO2 gas excretion in rabbit lungs. The shear stress stimulation produced CO2 gas in cultured human endothelial cells of pulmonary arterioles via the activation of F1/Fo ATP synthase. To confirm the findings in human subjects undergoing the operation with heart–lung machines, we aimed to evaluate the effects of a stepwise switch, from a partial to a complete cardiopulmonary bypass, of the circulatory blood volume (BV, 100% = 2.4 × cardiac index), on the end-expiratory CO2 pressure (PetCO2), maximal flow velocity in the pulmonary artery (Max Vp), the inner diameter (ID) of pulmonary artery, pulmonary arterial CO2 pressure (P mix v CO2), pulmonary arterial O2 pressure (P mix v O2), hematocrit (Hct), pH, the concentration of HCO3−, and base excess (BE) in mixed venous blood in 9 patients with a mean age of 72.3 ± 3.4 years. In addition, the effects of the decrease in Hct infused with physiological saline solution (PSS) on PetCO2 were investigated in the human subjects. An approximately linear relationship between the PetCO2 and Max Vp was observed. The pumping out of 100% BV produced little or no change in the Hct, pH, P mix v CO2, and P mix v O2, respectively. The hemodilution produced by intravenous infusion of PSS caused a significant decrease in the Hct, but not in the PetCO2. In conclusion, another route of CO2 gas excretion, independent of red blood cells, may be involved in human lungs.

Mesenteric lymph has received attention for its important roles in nutrient absorption and mucosal immunity. It contains cytokines and immune cells that control the immune responses. The aim of this study to evaluate the effects of cocoa butter intake on rat mesenteric lymph flow, cell density, and the IL-1β, IL-6 and IL-10 levels in the lymph. Cocoa butter intake increased the lymph flow, cell density and the cytokine levels in the lymph. Clodronate released IL-1β, IL-6 and IL-10 and slightly accelerated the cocoa butter intake-mediated release of IL-6, but not that of IL-1β and IL-10. A Toll-like receptor (TLR) 2 and 4 inhibitor significantly reduced the linoleic acid-mediated releases of IL-1β, IL-6 and IL-10 in the medium of isolated cells from lamina propria of jejunal villi. Our findings suggest that cocoa butter intake regulates gut immunity through the release of IL-1β, IL-6 and IL-10 via a negative feedback mechanism.

We previously demonstrated that water intake increased mesenteric lymph flow and the total flux of IL-22 in rat jejunum. The drained water and the higher permeability of albumin in the jejunal microcirculation contributed to increase the lymph flow and IL-22 transport via the activation of great bulk flow in the jejunal villi. To address the effects of water intake-mediated great bulk flow-dependent mechanical force on jejunal physiological function and immunological regulation of innate lymphoid cells (ILC)-3, we examined the effects of shear stress stimulation on cultured rat myofibroblast cells. Next, we investigated the effects of water intake on podoplanin and IL-22 expressions in cultured human intestinal epithelial cells and rat in vivo jejunal preparations, respectively. Shear stress stimulation of the myofibroblast cells induced ATP release via an activation of cell surface F1/F0 ATP synthase. ATP produced podoplanin expression in the intestinal epithelial cells. Water intake accelerated immunohistochemical expressions of podoplanin and IL-22 in the interepithelial layers and lamina propria of the jejunum. ATP dose-dependently increased IL-22 mRNA expression in ILC-3, which are housed in the lamina propria. Water intake also increased immunohistochemical and mRNA expressions of ecto-nucleoside triphosphate diphosphohydrolases 2 and 5 in jejunal villi. In conclusion, water intake-mediated shear stress stimulation-dependent ATP release from myofibroblast cells maintains higher tissue colloid osmotic pressure in the jejunal microcirculation through podoplanin upregulation in the interepithelial layers. ATP induces IL-22 mRNA expression in ILC-3 in jejunal villi, which may contribute to regulation of mucosal immunity in small intestine.NEW & NOTEWORTHY We investigated effects of shear stress stimulation on cultured myofibroblast cells and water intake on podoplanin and IL-22 expressions in rat jejunal villi. The stimulation induced ATP release from the cells. Water intake accelerated podoplanin and IL-22 expression levels. ATP increased IL-22 mRNA expression in innate lymphoid cells (ILC)-3. Hence, water intake maintains higher osmotic pressure in the jejunal villi through ATP release and podoplanin upregulation. Water intake may regulate the mucosal immunity.

The higher permeability of the venules in jejunal microcirculation to albumin contributes to the increased mesenteric lymph formation. Recently, we demonstrated that water intake induced serotonin release from enterochromaffin cells in rat jejunum, serotonin of which circulated through the portal vein into blood circulation and then increased the mesenteric lymph formation. The mode of action of serotonin remains unclear. Therefore, we aimed to clarify the mechanisms involved in the regulation of the jejunal lymph formation with permeant albumin in in vivo rat experiments. We investigated the effects of intravenous administration of serotonin or water intake on the jejunal-originated lymph volume and the concentration of albumin in the lymph in the presence or absence of L-NAME. The effects of intravenous administration of L-NAME, nicardipine, A23187, and ML-7 on the lymph formation with permeant albumin were also evaluated. Serotonin or water intake significantly increased the mesenteric lymph volume with permeant albumin in the jejunal microcirculation. The serotonin- and water intake-mediated responses were significantly reduced by the pretreatment with intravenous administration of L-NAME. Intravenous administration of L-NAME itself also decreased significantly the jejunal lymph formation. Administration of A23187 and ML-7 significantly reduced the jejunal lymph formation with permeant albumin. In contrast, administration of nicardipine significantly increased the lymph formation. In conclusion, portal venous blood flow- or serotonin-mediated NO release from venular endothelial cells plays physiologically key roles in the lymph formation in rat jejunum via the extrusion of calcium ions and inactivation of MLCK in endothelial cells.

In this review, with our current studies we demonstrated medical evidence that water and food intake are useful for IL-22-related mucosal immunity-dependent maintenance of health care. The traditional Japanese health care practices recommend daily consumption of suitable volume of water. However, immunological mechanisms that support of the traditional practices are still unsolved. We focused on type 3 innate lymphoid cells (ILC3s), because the ILC3s are mainly housed in the lamina propria of the jejunum. IL-22 released from the ILC3 is transported through mesenteric lymph in collaboration with the albumin-mediated movement of consumed water. Thus, water intake-mediated upregulation of IL-22-dependent mucosal immunity contributes to the traditional Japanese health care practices. We also reviewed current studies that food intake-mediated increase in VIP-dependent neuronal activity in the small intestine and the food intake included with tryptophan-derived metabolites may accelerate the IL-22 in ILC3s-dependent mucosal immunity and then contribute in keeping health care.

The present study aims to investigate the roles of water intake in serotonin production and release in rat jejunum. We evaluated the changes in concentrations of serotonin in the portal vein and mesenteric lymph vessel induced by the intragastric administration of distilled water. The density of granules in enterochromaffin cells and the immunoreactivity of serotonin in the jejunal villi were investigated before and after water intake. The effects of intravenous administration of serotonin and/or ketanserin on mesenteric lymph flow and concentrations of albumin and IL-22 in the lymph were also addressed. Water intake increased serotonin concentration in the portal vein, but not in the mesenteric lymph vessel. The flux of serotonin through the portal vein was significantly larger than that through the mesenteric lymph vessel. Water intake decreased the density of granules in the enterochromaffin cells and increased the immunoreactivity of serotonin in the jejunal villi. The intravenous administration of serotonin increased significantly mesenteric lymph flow and the concentrations of albumin and IL-22; both were significantly reduced by the intravenous pretreatment with ketanserin. We showed that serotonin released from enterochromaffin cells by water intake was mainly transported through the portal vein. Additionally, serotonin in blood was found to increase mesenteric lymph formation with permeant albumin in the jejunal villi via the activation of 5-HT2 receptor.

We demonstrated pulmonary arteriolar blood flow-mediated CO

The present study aims to investigate the roles of water intake in serotonin production and release in rat jejunum. We evaluated the changes in concentrations of serotonin in the portal vein and mesenteric lymph vessel induced by the intragastric administration of distilled water. The density of granules in enterochromaffin cells and the immunoreactivity of serotonin in the jejunal villi were investigated before and after water intake. The effects of intravenous administration of serotonin and/or ketanserin on mesenteric lymph flow and concentrations of albumin and IL-22 in the lymph were also addressed. Water intake increased serotonin concentration in the portal vein, but not in the mesenteric lymph vessel. The flux of serotonin through the portal vein was significantly larger than that through the mesenteric lymph vessel. Water intake decreased the density of granules in the enterochromaffin cells and increased the immunoreactivity of serotonin in the jejunal villi. The intravenous administration of serotonin increased significantly mesenteric lymph flow and the concentrations of albumin and IL-22; both were significantly reduced by the intravenous pretreatment with ketanserin. We showed that serotonin released from enterochromaffin cells by water intake was mainly transported through the portal vein. Additionally, serotonin in blood was found to increase mesenteric lymph formation with permeant albumin in the jejunal villi via the activation of 5-HT

The traditional Japanese health care custom recommends that a suitable volume of water is consumed. However, physiological and immunological mechanisms in support of this practice are unknown. Therefore, we conducted rat and rabbit in vivo experiments to investigate the effects of intragastric administration of distilled water on the jejunal-originated lymph flow and the concentrations and total flux of cells, albumin, long-chain fatty acids, and innate lymphoid cell 3 (ILC-3)-secreted interleukin-22 (IL-22) through mesenteric lymph vessels. The distribution and activity of ILC-3 in rat small intestine by water intake were evaluated using flow cytometry and RT-PCR. The intragastric administration of distilled water caused significant increases in rat mesenteric lymph flow and in the total flux of cells, albumin, long-chain fatty acids, and IL-22 through the lymph vessels. Intravenously injected Evans blue dye was rapidly transported into rabbit mesenteric lymph vessel and cisterna chyli. The distribution of ILC-3 and the expression of IL-22 mRNA were maximal in the lamina propria cells of the rat jejunum. No significant presence of ILC-3 in the lymph was observed in the control and under water intake conditions. In conclusion, the absorbed water in the jejunum is transported through mesenteric lymph vessels. The higher permeability of albumin in the jejunal microcirculation may play key roles in the transport of consumed water and the reservoir and transporter of long-chain fatty acids. Water intake also accelerates the transfer of IL-22 to the mesenteric lymph, which may contribute, in part, to maintaining and promoting the innate immunity in the body. NEW & NOTEWORTHY The higher permeability of albumin-mediated transport of water-soluble substances in mesenteric lymph vessels of the jejunum may have a large impact on the classic concept suggesting that water-soluble small molecules travel to the liver via the portal vein. ILC-3 is mainly housed in the lamina propria of the jejunum, especially its upper part. IL-22 released from the ILC-3 is also transported through mesenteric lymph in collaboration with the albumin-mediated movement of consumed water.

We created a driving simulation test for assessing drivers’ hazard-perception skills by measuring their palmar sweating response (PSR), skin potential reflex (SPR), and ability to operate the steering wheel, accelerator, and brake. We recruited 52 elderly people who were aged ≥60 years and were active drivers. These participants undertook the driving simulation test, and we measured their PSR, SPR, and device operation responses. PSR tended to be greater in hazard scenes that involved anticipation or complexity processes, suggesting the involvement of factors such as the participants’ readiness to anticipate hazards and their emotional changes. SPR response was faster in hazard scenes that involved a surprise process. SPR response was faster than braking response, except in scenes where the participants had only a fleeting moment to react to the hazard, suggesting that SPR is a suitable evaluation index for hazard perception timing. Two participants were suspected of having decreased cognitive function according to the Mini-Mental State Examination. These individuals exhibited PSR and SPR responses that were not consistent with the hazard scenes, and they made errors in their operation of the driving simulator. These findings highlight the need to examine the impact of decreased cognitive function on hazard perception.

Background: Previous animal studies have shown that intragastric administration of water can accelerate mesenteric lymph flow. Similarly, human studies have shown that abdominal breathing can induc...

In this chapter, we focus on the physiology of gastrointestinal lymphatic system and then demonstrate with current inspired studies that (1) functional properties of the intestinal microcirculation are summarized as higher permeability of plasma protein, especially albumin through the venule walls, which contribute to the oncotic pressure-mediated much absorption of interstitial fluid into lymphatic capillaries, (2) the larger amount of lymph formation in the intestinal villi may be related to the presence of lacteal vessels in the tissues, (3) the mesenteric collecting lymphatics demonstrate marked heart-like spontaneous contractions working as transport of the larger amount of lymph to chylous cyst, (4) the sentinel lymph node (SLN) may be defined as the node subjected to higher lymph flow from physiological point of view, and (5) the higher lymph flow may be, in part, related to develop the suitable microenvironment of the SLN for metastasis of carcinoma cells, which is produced by the cell surface F1/F0 ATP synthase-dependent overexpression of intercellular adhesion molecule-1 on the marginal endothelial-like cells of SLN.

To confirm our previous study that abdominal respiration has induced hemodilution in human subjects, we performed in-vivo experiments involving anesthetized rabbits. Fifteen 6- to 7-month-old male Japanese white rabbits were used in the animal experiments. Anesthesia was maintained with 2.5%-3.0% isoflurane under N

OBJECTIVE We studied the effects of S1P on the diameter and spontaneous contraction of murine iliac collecting lymph vessels. METHODS The isolated lymph vessel was cannulated with two glass micropipettes and then pressurized to 4 cmH(2) O at the intraluminal pressure. The changes in lymph vessel diameter were measured using a custom-made diameter-detection device. Immunohistochemical studies were also performed to confirm S1P receptors on the lymph vessels. RESULTS S1P (10(-7) M) had no significant effect on the frequency or amplitude of the lymph vessels' spontaneous contractions. In contrast, S1P (10(-8) -10(-6) M) produced a concentration-related reduction in lymph vessel diameter (tonic contraction). Pretreatment with 10(-4) M l-NAME or 10(-5) M aspirin had no significant effect on the S1P-induced tonic contraction of the lymph vessels. To evaluate the intracellular signal transduction pathway responsible for the S1P-induced tonic contractions and their Ca(2+) -dependence, we investigated the effects of JTE013, VPC23019, U-73122, xestospongin C, and nifedipine on the S1P-induced tonic contractions. All of these inhibitors except VPC23019 and nifedipine significantly reduced the S1P-induced tonic contractions. S1P (5x10(-7) M) also induced significant tonic contractions in the lymph vessels that had been superfused with high K(+) Krebs-bicarbonate solution or Ca(2+) -free high K(+) Krebs solution containing 1 mM EGTA. S1P2 receptors were immunohistochemically detected in the lymph vessels. CONCLUSION These findings suggest that neither endogenous NO nor prostaglandins are involved in the S1P-induced tonic contraction of lymph vessels, which is mainly caused by Ca(2+) release from intracellular Ca(2+) stores through the activation of S1P2 and 1,4,5 IP(3) receptors.

We examined the immunohistochemical properties of selective lymph vessel markers such as LYVE-1, podoplanin, Prox-1, and VEGF R3, as well as NO synthase (NOS) and cyclo-oxygenase (COX) in two kinds of human lymphatic endothelial cell isolated from collecting and initial lymph vessels. The constitutively expressed genes in the two kinds of lymphatic endothelial cell were also evaluated using oligonucleotide microarray analysis and RT-PCR. We also investigated the effects of the oxygen concentration in culture conditions on the proliferative activities of the two kinds of human lymphatic endothelial cell. Immunoreactivity to LYVE-1 and the RT-PCR expression level of LYVE-1 mRNA in endothelial cells of initial lymph vessels were stronger than those of collecting lymph vessels. Immunoreactivity to ecNOS, iNOS, COX1, and COX2 was also found to be significantly higher than in collecting lymph vessels. In contrast, an increase in the O2 concentration ranging from 5% to 21% caused a significant reduction in the proliferative activity of endothelial cells in collecting lymph vessels. In conclusion, these findings suggest that there exists a marked heterogeneity in the immunohistochemical, genomic, and proliferative activity of human lymphatic endothelial cells between initial and collecting lymph vessels. (*English Translation of J Jpn Coll Angiol, 2008, 48: 125-130.).

The contrast-enhanced ultrasound (CEUS)-guided method in combination with Sonazoid has not been clinically or experimentally evaluated with regard to its use for identifying sentinel lymph node (SLN) in the stomach. Therefore, we attempted to evaluate the usefulness of the CEUS-guided method with Sonazoid for imaging of the lymphatic channels and SLN of the stomach in a porcine model by comparing it with the conventional Evans blue dye-guided method. Twenty-eight 2 to 3-month-old swine weighing 17-30 kg were used in this experiment. Anesthesia was maintained with 2.0-3.0% isoflurane/O(2) inhalation. Sonazoid was injected into the intra-and sub-mucosal layers of the stomach. The intragastric or transcutaneous CEUS-guided method was used to identify the lymphatic channels and SLN of the stomach. Contrast imaging using the CEUS-guided method with Sonazoid enabled us to produce clear images of the afferent lymph vessel and SLN of the stomach until 2 h after the injection of Sonazoid. In addition, intranodal flow of the microbubble agent could be clearly identified using tissue linear harmonic images of the SLN. The SLN detection rate was not significantly different between the CEUS- and dye-guided methods. However, the Evans blue dye flowed out quickly (similar to 15 min after the injection) through the true SLN into the next LN of stomach. In conclusion, the use of the CEUS-guided method with Sonazoid might be the most useful clinical procedure for producing real-time images of the SLN of the stomach, and the linear harmonic images are also useful for evaluating intranodal structure within the SLN. (Cancer Sci 2011; 102: 2073-2081), Article, CANCER SCIENCE. 102(11):2073-2081 (2011)

We studied the effects of PDGF-BB on changes in the diameters of murine lymph vessels with or without intact endothelium. PDGF-BB induced dilation of the lymph vessels with endothelium. Pretreatment with l-NAME or removal of the endothelium caused a significant attenuation in the PDGF-BB-induced dilation. PDGF-BB also produced dose-related reduction of the diameters of the lymph vessels without endothelium. To evaluate intracellular signal transduction and Ca(2+) -dependence of the PDGF-BB-induced tonic contraction, we investigated the effects of imatinib, GW5074 (an inhibitor of Raf-1 kinase), U-73122 (an inhibitor of phospholipase C), and xestospongin C on the PDGF-BB-induced reduction responses. All of these inhibitors caused a significant attenuation in the PDGF-BB-induced reduction response that was significantly decreased by treatment with Ca(2+) -free Krebs-bicarbonate solution or nifedipine. Higher concentrations of PDGF-BB produced a marked reduction of lymph vessel diameter within both high K(+) Krebs-bicarbonate solution and Ca(2+) -free high K(+) Krebs solution containing 1mM EGTA. These findings suggest that PDGF-BB induced endothelium-dependent NO-mediated relaxation of lymphatic smooth muscles in murine lymph vessels. PDGF receptor β-mediated tonic contraction of the muscles through increased Ca(2+) influx through the membrane and the release of membrane-bound and intracellular Ca(2+) .

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/cm2 increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from 10−9 to 10−6 M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by 10−6M ATP was significantly reduced by 10−5 M suramin. Suramin (10−5 M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, 10−4 M apamin, 10−9 M iberiotoxin, 10−5 M 2-aminoethoxydephenyl borate, or 10−5M xestospongin C, but not 10−5 M glybenclamide or 10−5 M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small- (SKCa) and big-conductance (BKCa) Ca2+-activated K+ channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphate-mediated release of intracellular Ca2+ ions and the activation of Ca2+-activated K+ channels in LEC.

The effects of short-term simulated microgravity on the lymph dynamics of rat lymph nodes were investigated using a combination of Bollman's cage and head-down tilt (HDT). Efferent lymphatics of the iliac and mesenteric lymph nodes were cannulated for the collection of lymph. There was no significant difference in lymph flow rate from the iliac lymph nodes between non-HDT (control) and HDT rats. Lymph flow rate from the mesenteric lymph nodes in HDT rats was slightly higher than that obtained with the control. The cell count obtained from the iliac lymph nodes in HDT rats was significantly larger than those of the controls, while no significant difference in the number of cells from the mesenteric lymph nodes was observed between the control and HDT groups. The cells from the iliac lymph nodes in the control and HDT rats were mostly lymphocytes. The distribution of subsets of lymphocytes (CD3+, CD4+, CD8a+, and CD45R+) from the iliac lymph nodes in HDT rats was not significantly different from the subsets of lymphocytes in the control. Immunization did not affect the distribution of lymphocyte subsets from the iliac lymph nodes in the control and HDT groups. There was no significant difference in the concentrations of lymph albumin in iliac afferent or efferent lymphatics between the control and HDT groups. These findings suggest that HDT posture in Bollman's cage induces transient output of lymphocytes from the iliac lymph nodes of rats in vivo without changing the flow rate, lymphocyte subsets, or concentration of albumin.

The immunohistochemical properties of selective lymph vessel markers, and NO synthase (NOS) and cyclo-oxygenase (COX) activities, were examined in two kinds of human lymphatic endothelial cells isolated from collecting (macro-) and initial (micro-) lymph vessels. The constitutively expressed genes in the two kinds of lymphatic endothelial cells were also evaluated by using oligonucleotide microarray analysis and RT-PCR. We also investigated the effects of oxygen concentration in culture conditions or growth factors such as basic fibroblast growth factor (bFGF), VEGF-A, and VEGF-C on proliferation activities of the two kinds of human lymphatic endothelial cells. Immunoreactivity to LYVE-1 and the RT-PCR expression level of LYVE-1 mRNA in endothelial cells of micro-lymph vessels were stronger than those of macro-lymph vessels. Immunoreactivity to VEGF R1 was also observed as significantly stronger in the micro-lymph vessels. In contrast, the immunoreactivity to Prox-1 and the RT-PCR expression level of Prox-1 mRNA in endothelial cells of macro-lymph vessels were stronger than those of micro-lymph vessels. Similarly, immunoreactivity to ecNOS, iNOS, COX1, and COX2 was also found as significantly higher than in macro-lymph vessels. In contrast, the increase of O(2) concentration ranging from 5% to 21% caused a significant reduction of the proliferation activity of endothelial cells in macro-lymph vessels. In conclusion, these findings suggest marked heterogeneity in the immunohistochemical, genomic, and proliferation activity of human lymphatic endothelial cells between micro-(initial) and macro-(collecting) lymph vessels.

Stromal cells, together with extracellular matrix components, provide a tumor microenvironment that is pivotal for cancer cell growth and progression. In our previous study using a conditional transgenic mouse model of breast cancer, the overproduction of hyaluronan, a major extracellular constituent, accelerated tumor angiogenesis through stromal cell recruitment. This finding led us to investigate the role of hyaluronan in the lymphatic vessel system. Here, we have found that microenvironmental hyaluronan promoted tumor lymphangiogenesis concurrently with the formation of stromal structures. Additionally, lymphatic vessels frequently penetrated and accumulated into stromal compartments, and up-regulation of vascular endothelial growth factor-C and -D was detected at tumor-stromal interfaces. To assess the contribution of stromal cells to lymphangiogenesis in vivo, we established tumor-associated fibroblasts from hyaluronan-overproducing mammary tumors and implanted them together with carcinoma cells from control tumors or MCF-7 human breast carcinoma cells in nude mice. Carcinoma cells grew rapidly in association with marked stromal reactions and lymphangiogenesis. Without the stromal cells, however, the tumors developed slowly with less stroma and lymphatic vessels. These findings underline the significance of tumor-associated stroma in the promotion of intratumoral lymphangiogenesis and suggest a pivotal role for the hyaluronan-rich tumor microenvironment.

We investigated the pivotal roles of glucose and its transporter in the regulation of mechanical activity of isolated rat thoracic ducts and then examined whether mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) are involved in those responses. In the absence of extracellular glucose, the thoracic ducts showed pump activity during 120 min. Extracellular glucose caused a dose-dependent increase in the frequency of pump activity and a constriction in the thoracic ducts. Pump activity of the thoracic ducts in 0 mm glucose was completely inhibited in the presence of chlorogenic acid (an inhibitor of glucose-6-phosphatase). Cytochalasin B, an inhibitor of facilitative glucose transporter (GLUT), or phlorizin, an inhibitor of sodium-dependent glucose cotransporter (SGLT), significantly reduced the frequency of pump activity and dilated the thoracic ducts. A decrease in the frequency of pump activity induced by 5-hydroxydecanoate (5-HD, a selective blocker of mitoK(ATP)) was completely reversed by ruthenium red (an inhibitor of Ca(2+) uniporter in mitochondria). Diazoxide (a selective opener of mitoK(ATP)) significantly increased the frequency of pump activity. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, a protonophore of mitochondrial proton pump action) significantly reduced the frequency of pump activity and dilated the thoracic ducts. Collectively, these findings suggest that glucose derived from intracellular glycogen and/or through GLUT/SGLT in lymphatic smooth muscles contributes to the regulation of the pump activity of isolated rat thoracic ducts, and that mitoK(ATP) in the cells may partially serve as a modulator of the mechanical functions associated with mitochondrial Ca(2+) uptake.

We investigated the effects of NT-702, a selective phosphodiesterase (PDE) 3 inhibitor, on arterioles isolated from rabbit lumbar spinal cords. NT-702 caused a dose-dependent dilation of the isolated spinal arterioles. The disruption of endothelium produced a significant reduction of higher concentrations (10(-7) and 10(-6) M), but not lower concentrations (less than 10(-8) M), of NT-702-induced vasodilation. The NT-702-induced vasodilation of the arterioles with endothelium was not affected by pretreatment with an inhibitor of nitric oxide, cyclooxygenase, or cytochrome P-450 monooxygenase. In contrast, catalase reduced significantly the higher concentrations of NT-702-induced vasodilation only. Tetraethylammonium (TEA) completely reduced the lower concentrations of NT-702-induced vasodilation, but decreased only partially the higher concentrations of NT-702-induced vasodilation of the arterioles with endothelium. Hydrogen peroxide dilated significantly the isolated arterioles with endothelium, the response of which was reduced significantly by TEA. KT5720 (a selective protein kinase inhibitor) significantly decreased both the lower and higher concentrations of NT-702-induced vasodilation of the arterioles with endothelium. The findings suggest that NT-702 dose-dependently dilated the isolated spinal arterioles of rabbits via endothelium-dependent and endothelium-independent mechanisms. Protein kinase A (PKA)- and TEA-sensitive K(+) channels may be involved in the NT-702-induced vasodilation. Moreover, hydrogen peroxide may contribute in part to the endothelium-dependent higher concentrations of NT-702-induced vasodilation.

We successfully isolated human lymphatic endothelial cells from afferent lymph vessels (HALEC) of sentinel lymph nodes in patients with breast cancer by using trypsin digestion. The cells were cultured in EGM-2 medium with 10% FBS under the condition of 5% O2, 5% CO2, and 90% N2 at 37 degrees C. The cultured cells exhibited a monolayer with cobblestone appearance and a marked phagocytosis of Dil-Ac-LDL. Immunohistochemical lymphatic vessel markers were also found, such as podoplanin, LYVE-1, VEGF receptor 3, and Prox-1. Quantitative RT-PCR analysis also showed that podoplanin, VEGF R3, and Prox-1 mRNA were expressed more selectively in the cultured cells. The cells had marked immunoreactivity to antisera of ecNOS, iNOS, COX1, and weak reactivity of COX2. Constitutively expressed cell-type specific genes of the cultured cells were also analyzed by oligonucleotide microarray methods. Compared with human umbilical vein endothelial cells (HUVEC), the cells selectively expressed 88 known genes such as angiopoietin-like 4, oxygen radicals-related enzymes, and adhesion molecules and the related proteoglycans. The findings suggest that the cultured cells seem to be human lymphatic endothelial cells. In conclusion, the isolated, cannulated and enzymatic digested method we adopted for culture of human lymphatic endothelial cells may be easy and useful for investigating cellular, molecular biological, and genomic properties of the cells.

Lymphatic metastasis to the regional lymph nodes through the lymphatic vessels is an important indicator of poor prognosis in many types of malignant tumors. Recently, much attention has been paid to lymphangiogenesis for its possible role on tumor progression in various carcinomas. However, morphological evidence that lymphatic vessels actively proliferate in colorectal carcinoma has not been reported. Here, we first devised a triple immunostaining method to detect proliferating lymphatic vessels utilizing antibody to Ki-67 antigen as a marker of cell proliferation, antibody to cytokeratin as an epithelial cell marker, and antibody to podoplanin as a lymphatic vessel-specific marker. Ki-67/podoplanin-immunoreactivity enabled us to identify proliferating lymphatic vessels, while cytokeratin immunoreactivity allowed us to distinguish proliferating lymphatic vessels from Ki-67/cytokeratin-positive carcinoma cells in lymphatic lumens. Analyzing 64 colorectal carcinoma patients' samples using this technique, we showed that both lymphatic vessel density and proliferating activity of lymphatic vessels were significantly increased in colorectal carcinoma tissues compared with their normal counterparts. We then examined the correlation between the degree of lymphangiogenesis and patients' prognosis or clinicopathological variables, but no statistically significant differences were obtained in these analyses. Thus, these results combined together indicate that extensive lymphangiogenesis occurs in colorectal carcinoma, but that the degree of lymphangiogenesis alone is not an independent prognostic factor for this disease.

To establish effective lymph drainage methods and develop concise and accurate clinical techniques for evaluating lymph drainage in healthy individuals and patients with cancer treatment-related lymph edema, we investigated the numbers of red (RBC) and white (WBC) blood cells, and platelets (PLT) in blood, hematocrit (Ht), and the blood concentrations of total protein (TP), albumin (Alb), and anti-diuretic hormone (ADH) before and after 5 min manual lymph drainage, followed by 30 min rest with or without abdominal respiration in the supine or sitting position in 48 healthy volunteers. The 5 min facial, upper and lower extremities lymph drainage, followed by 30 min rest in the supine position induced significant reductions of the TP and Alb in all subjects, and their RBC and Ht levels in some subjects. The 30 min rest only in the supine position without lymph drainage produced also significant reductions of blood TP and Alb. In addition, abdominal respiration in the supine position without manual lymph drainage caused more significant hemodilution, being significant reductions of TP, Alb, RBC, Ht, and ADH in all volunteers. These findings may be related to effective lymph drainage from the chylocyst. Furthermore, it also resulted in a significantly increased micturition desire. In conclusion, abdominal respiration during 30 min rest in the supine position is effective at inducing lymph drainage, and the associated induction of hemodilution and lowering of the blood ADH concentration (and increased micturition desire in some cases) can be used to accurately assess the extent of lymph drainage.

This study was performed to investigate the heterogeneity of physiological and pharmacological properties in segments of the facial veins with special reference to selective brain cooling. Canine facial veins were isolated and the isometric tension of each segment was measured using the organ bath technique. Vessels in the segments of the facial veins that run opposite to the buccal cavity automatically produced myogenic tone and tended to show spontaneous contractions, but vessels in other segments did not. When no contractile agent was used for precontraction, noradrenaline and adrenaline produced dose-dependent relaxations in the former venous segments, but contractions in the latter ones. A Schild plot analysis for metoprolol against denopamine and for ICI118,551 against salbutamol showed that the venous segments running opposite the buccal cavity contained both beta(1)- and beta(2)-adrenoceptors, but the other venous segments contained only beta(2)-adrenoceptors. Electrical field stimulation-induced tetrodotoxin-sensitive relaxations in the former venous segments were diminished by pretreatment with metoprolol, but not with ICI118,551, indicating that the electrical stimulation-induced relaxation may be related to the activation of beta(1)-adrenoceptors in the venous smooth muscles. In conclusion, the heterogeneity of the functional properties, especially in the distribution of beta-adrenoceptors, in different segments of canine facial veins was observed in the present study, and autoregulatory mechanisms, humoral mechanisms, and neural mechanisms were suggested to affect cranial venous drainage.

We investigated the effects of cilostazol, a potent inhibitor of cGMP-inhibited cAMP phosphodiesterase, on mechanical activity of isolated pressurized rabbit cerebral penetrating arterioles with special reference to the function of the endothelium. Both cilostazol and milrinone, another inhibitor of cAMP phosphodiesterase, produced vasodilation of the cerebral penetrating arterioles in a dose-dependent manner. Pretreatment with selective inhibitors of cyclooxygenase or nitric oxide synthase, or chemical denudation of the endothelial cells caused no significant effect on the cilostazol-mediated vasodilation of the cerebral arterioles. A selective large-conductance calcium-activated potassium channel inhibitor, iberiotoxin, and a selective protein kinase A inhibitor, H-89, caused no significant effect on the cilostazol-mediated vasodilation. In the cerebral arterioles, low concentration (10–6M) of cilostazol or milrinone caused a significant shift of the dose-vasodilatory response curve for adenosine to the left. These findings suggest that cilostazol produces vasodilation independent of the presence of the endothelium or activation of endogenous vasodilative prostaglandins, nitric oxide, calcium-activated potassium channel and protein kinase A. In conclusion, the vasodilator action of cilostazol may, in part, contribute to the beneficial effect of preventing lacunar cerebral infarction in patients with functional damage of the endothelium in cerebral penetrating arterioles.

There is no reported study in which the lymphatic flow pathway in lymph nodes has been visualized and investigated in in vitro studies. The purpose of the present study is first to develop an isolated rat lymph node preparation circulated through the afferent and efferent lymph vessels in order to visualize lymphatic flow pathway within the node by using a video microscope system, and then to evaluate lymphatic flow dynamics by using fluorescence-labeled substances.In the present study, rat iliac lymph node was isolated. The afferent and efferent lymph vessels of the lymph node were cannulated with glass micropipettes in an organ chamber, while the small-sized arteries and veins connected to the lymph node were secured with sutures. Krebs-bicarbonate solution with or without fluorescent probes [FITCdextran, mol. weight; 77,000 and/or fluoresbrite carboxylate-microspheres (FC-microspheres) mean diameter of 1 microm] was circulated through the lymph vessels of the node. The time-dependent lymphatic pathway of fluorescent probes was investigated with a video microscope system. FTIC-dextran first spread through the cortex and subsequently reached the medulla of the lymph node. A follicle-like structure became evident in the cortex. FITC-dextran appeared in the efferent lymph vessel at 109 +/- 21 sec after its circulation began, while FC-microspheres distributed in the lymph node did not emerge from the node within 20 min after their introduction.In vitro preparations constructed in the present study will enable us to visualize the lymphatic flow pathway of fluorescent substances within the isolated iliac lymph nodes of rats in the absence of blood circulation.

Flow-induced vasodilation in hamster cremasteric arterioles was investigated with special reference to the roles of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Arterioles (∼60 µm resting diameter) were cannulated, and suffused with MOPS solution at 37°C (mean intraluminal pressure: 80 cm H2O). Step increases in the perfusate flow elicited a dose-dependent vasodilation, which was almost proportional to the increases in calculated wall shear stress (WSS). Nω-nitro L-arginine methyl ester (L-NAME, 100 µM) reduced the flow-induced vasodilation by ∼50%, whereas indomethacin (10 µM) produced no significant effect. In the presence of L-NAME, the residual vasodilation was eliminated by treatment with the cytochrome P-450 monooxygenase inhibitor 17-octadecynoic acid (17-ODYA, 50 µM), sulfaphenazol (10 µM), tetraethylammonium (TEA, 3 mM; a nonselective Ca2+-activated K+ channel inhibitor), or charybdotoxin (ChTX, 0.1 µM; intermediate or large conductance Ca2+-activated K+ channel inhibitor). In the absence of L-NAME, the dilation was also reduced by ∼50% by treatment with 17-ODYA, TEA, or ChTX. The residual vasodilation was eliminated by additional treatment with L-NAME. The inhibitor of ATP-sensitive K+ channels (KATP), glibenclamide, also caused a significant, but partial, reduction of the flow-induced vasodilation. The residual vasodilation was completely reduced by additional treatment with 17-ODYA, but not L-NAME. These findings suggest that in hamster cremaster, higher flow rate produces NO, KATP, and EDHF vasodilation of the arterioles under physiological conditions.

A general formula (Oka and Azuma's equation) has been rigorously derived for the circumferential wall tension in a hollow cylindrical tube in equilibrium. To evaluate the validity and usefulness of Oka and Azuma's equation, T = P(1) x r(1) - P(2) x r(2) (T, circumferential wall tension; P(1) and P(2), internal and external pressures of the tube; r(1) and r(2), the corresponding internal and external radii), we experimentally investigated changes in circumferential wall tension of noninjured (control) and injured dog common carotid arteries by using a newly developed apparatus with a photo- and X-ray-sensitive image sensor. We also studied histological features of the control and injured arteries with special reference to the relation of biomechanical properties. Two types of animal models with injured arteries--balloon-induced neointima or external collar-induced intimal thickening--were adopted in the present study. In the control arteries, the circumferential wall tension was experimentally confirmed to change from negative to positive by an increase in intraluminal pressure ranging from 50 to 180 mmHg. The critical intraluminal pressure that produced 0 dyne/cm of the circumferential wall tension was around 135 mmHg. The activation of arterial smooth muscles caused a significant increase in the critical pressure in the control arteries. In the arteries injured by neointima, the critical intraluminal pressure was significantly lower than that in the control. The activation of smooth muscles also significantly increased the critical pressure in the injured arteries. Histological examination demonstrated the existence of a circumferential neointimal formation along with a shortening of the internal diameter. In other arteries injured by intimal thickening, the circumferential wall tension was always negative at intraluminal pressure ranging from 50 to 180 mmHg. Newly developed structures consisted of elastic and collagen fibers, smooth muscles, and extracellular matrix in the intima and media of the injured arteries. These experimental findings suggest that the circumferential wall tension of dog common carotid arteries has been confirmed experimentally to become negative. We have also concluded that circumferential wall tension calculated with Oka-Azuma's equation may be one of the best parameters for evaluating changes in the biomechanical and histological properties of pathologically injured arteries.

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.

We examined chronic effects of 17beta-estradiol (E(2)beta) on the responses of isolated rat anterior cerebral small arteries to vasoactive substances with special reference to endothelial function. Female Sprague-Dawley rats were separated into four groups: (1) sham-operated group (Sham), (2) sham-operated plus E(2)beta treated group (Sham+E), (3) ovariectomized group (OVX), (4) ovariectomized plus E(2)beta treated group (OVX+E). 5-Hydroxytryptamine (5-HT) (10(-10)-10(-3) M) and U46619 (10(-15)-10(-8) M) induced concentration-dependent contractions in the cerebral small arteries. The 5-HT- and U46619-induced contractions were not affected by pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME). No significant difference in high potassium (80 mM)- and the agonists-mediated contractions was observed among the four groups. Administration of acetylcholine (ACh) (10(-9)-10(-3) M) and sodium nitroprusside (SNP) (10(-8)-10(-3) M) caused dose-related relaxations in the cerebral small arteries precontracted by 10(-8) M U46619. Chronic treatment with E(2)beta caused a significant potentiation of the ACh-induced relaxations in the Sham+E and OVX+E groups. The dose-response curve for ACh in the OVX group was quite similar to that obtained with the Sham group. The ACh-induced relaxation was reduced significantly by pretreatment with 3 x 10(-5) M L-NAME, and an additional treatment with 10(-3) M L-arginine reversed significantly the L-NAME-induced inhibition. The removal of endothelial cells produced a significant reduction of the ACh-induced relaxation. Indomethacin (10(-5) M) did not alter the ACh-induced relaxation. The findings suggest that E(2)beta potentiates ACh-induced endothelium-dependent relaxation in rat anterior cerebral arteries and that the potentiation may be, in part, mediated by increasing production and release of endogenous NO from the endothelial cells.

We examined the responses of pressurized bovine retinal functional arterioles (97-185 microm in diameter and approximately 3 mm long) to vasoactive substances and the mode of action of acetylcholine (ACh) on the pressurized arterioles. The retinal arterioles were cannulated at both ends with glass micropipettes and perfused at a constant pressure of 60 mmHg. Vasoconstrictions of the retinal arterioles were induced by prostaglandin F(2 alpha) (PG F(2 alpha)), U46,619, noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in a dose-dependent manner. The decreasing order of potency (pD(2) value) in the constrictive responses was as follows: 5-HT = U46,619NAPG F(2 alpha). On the other hand, sodium nitroprusside (SNP), isocarbacyclin (a stable prostaglandin I(2) analog), ACh, and isoproterenol (ISP) caused dose-dependent vasodilatation in the pressurized retinal arterioles preconstricted with high-potassium solution (40 mM K+). The decreasing order of potency in the vasodilative responses was as follows: isocarbacyclinSNPACh. The ACh-induced vasodilatation was suppressed significantly by pretreatment with N omega-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M). A treatment with L-arginine (10(-3) M) in the presence of 3 x 10(-5) M L-NAME reversed completely the L-NAME-induced reduction of the vasodilatation. These results suggest that ACh causes the production and release of endogenous nitric oxide or its related compounds, which results in vasodilatation of the pressurized bovine retinal functional arterioles.

We examined the effects of plasma on contractile responses of isolated dog external jugular veins to a thromboxane A(2) analog, U46619, and noradrenaline. Pretreatment with 1.0% plasma in Krebs-bicarbonate solution, but not 0.1%, caused a significant left and upward shift in the concentration-contractile response curve for U46619. The plasma-mediated potentiation of the response to U46619 was found in the venous segments without intact endothelium. The administration of 2x10(-5) M lysophosphatidylcholine in Krebs-bicarbonate solution with no plasma also produced a significant left and upward shift of the concentration-contractile response curve for U46619, the shift being quite similar to that obtained with 1.0% plasma. In contrast, pretreatment with 1.0% plasma or 2x10(-5) M lysophosphatidylcholine produced no significant effect on the noradrenaline-mediated contractions in the venous segments. Pretreatment with 10(-4) M L-ascorbate or 0.1 mg/ml alpha-tocopherol in the presence of 1.0% plasma caused a significant reduction in the plasma-mediated potentiation of the contractile responses to U46619. These findings suggest that lysophosphatidylcholine, a major phospholipid component of oxidized low-density lipoproteins, may contribute, in part, to the plasma-mediated potentiation of contractile responses of the isolated veins to U46619, and that the antioxidant vitamin, L-ascorbate, or alpha-tocopherol significantly reduces the plasma-mediated potentiation of the contractile responses to U46619, which may be related to inhibiting the production of lysophosphatidylcholine in plasma.

Cilostazol, a potent inhibitor of guanosine 3':5'-cyclic monophosphate (cGMP)-inhibited adenosine 3':5'-cyclic monophosphate (cAMP) phosphodiesterase (PDE3), has been used clinically for the treatment of chronic peripheral arterial occlusive disease. The beneficial effect of cilostazol is attributed to both anti-platelet aggregating activity and vasodilation. However, the effect of cilostazol on resistance-sized vasculature is not well documented. Furthermore, mechanisms of vasodilation and influence on endothelium function are not fully understood. Thus, we investigated the vasodilator action of cilostazol using isolated, pressurized rabbit spinal arterioles with special reference to the functional endothelium. Cilostazol, acetylcholine (ACh), isocarbacyclin (prostacyclin analogue), and sodium nitroprusside (SNP) all produced concentration-dependent vasodilations of isolated spinal arterioles with endogenous myogenic tone. The order of potency of these agonists was isocarbacyclinAChSNPcilostazol. Indomethacin (10 micro M, a cyclo-oxygenase inhibitor), N(omega)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor, 30 micro M), or chemical denudation of the endothelial cells did not significantly alter the cilostazol-induced arteriolar dilation. Furthermore, stimulating the release of endothelium-derived relaxing factors by administering ACh (100 nM), or treating with isocarbacyclin (1 nM) or SNP (3 nM) did not significantly modify the cilostazol-induced vasodilation. These results suggest that cilostazol produces the vasodilation of isolated, pressurized rabbit spinal arterioles independent of the functional endothelium. We infer that the vasodilator action of cilostazol in the spinal arterioles may be attributed to a yet unknown mechanism that is independent of the PDE3 inhibition.

As one of the lymphatic functions, it is well known that the transport and drainage of hydrophilic substances including plasma protein through the lymphatic system play pivotal roles in maintaining the homeostasis of the internal environment between the cells in tissues in collaboration with the exchange of the substances through the blood capillaries and venules. The physiological functions of the lymphatic system have been studied by many investigations of microcirculation, i.e., Yoffey and Courtice, Ruszunyak et al., Foldie and Casley-Smigh et al., Roddie, Schmid-Schonbein et al., and Ohhashi et al. On the other hand, it is also well known that the initial clinical signs of primary diseases such as inflammation, tumors, and circulatory disorders including infarction and thrombosis appear as functional abnormalities of the internal environment in tissues. These abnormalities of the functions are strongly related to immunological defense reactions around the internal environment and abnormal actions of the transport and drainage of the lymphatic system. Taking into consideration the current inspired findings in lymphatic physiology, innate immunology, and oncology, we have proposed a new lymphology combined with new knowledge of the three above-mentioned academic fields from a defense mechanism points of view. In this review, we would like to demonstrate comprehensively our latest studies related to the possibility of establishing a new lymphology, hoping the readers will evaluate this possibility.

To address physiological and pathophysiological meanings of condensing effect of albumin in lymph through collecting lymph vessel walls, we established human lymphatic endothelial cells (LEC) and evaluated the size-dependent regulation of the permeability of such layers to hydrophilic substances. We also investigated the effects of tumor necrosis factor (TNF)-α or interleukin (IL)-1β on the permeability and on the morphology of human LEC. Significant amounts of 4 kDa dextran, but not 12 or 66 kDa dextran, passed through the layers. TNF-α or IL-1β induced significant increases in the permeability to 4 and 12 kDa dextrans. TNF-α or IL-1β also produced significant redistribution of the cytoskeletal F-actin in the LEC, which resulted in changes in their shape. Pretreatment with Y-27632, a Rho kinase inhibitor, or PD98059, an extracellular signal-regulated kinase (ERK) phosphorylation inhibitor, significantly abolished the TNF-α- or IL-1β-induced increases in the permeability of the layers to 4 and 12 kDa dextrans. Y-27632 and PD98059 significantly inhibited the changes in the F-actin distribution of the LEC produced by TNF-α or IL-1β. TNF-α or IL-1β caused significant increases in ERK 1/2 phosphorylation in the LEC, which were significantly inhibited by Y-27632 or PD98059. These findings suggest that the human LEC layer plays key roles in the transport of hydrophilic substances through collecting lymph vessel walls and that TNF-α or IL-1β significantly increases the permeability of the layers to 4 and 12 kDa dextrans via Rho kinase activation and the ERK 1/2 phosphorylation-mediated reorganization of F-actin in the LEC.