Fractional Pump Flow Research Articles

Cervical lymphatic vessel dysfunction induced by amyloid-β and in the 5x-FAD mouse model of Alzheimer’s disease

Alzheimer’s dementia is a multifactorial disease characterized by amyloid-β (Aβ) accumulation in the brain. Aβ clearance occurs partially via meningeal lymphatics into cervical lymph nodes through superficial cervical lymphatic vessels (sCLV), which relies on intrinsic contractions of sCLV. Previous studies showed increased Aβ build up in the brain following ablation of meningeal lymphatics; however, whether Aβ alters sCLV function remains unclear. We hypothesize that sCLV contractile function will be reduced by acute Aβ exposure and in the 5x-FAD mouse model of Alzheimer’s disease. Contractile function of freshly isolated sCLV was assessed using pressure myography. Acute Aβ exposure was achieved by intraluminal incubation of pressurized sCLV with Aβ(1-40) (5 μM) or vehicle for 15 minutes prior to the start of the experiment. Repeated end-diastolic (EDD) and end-systolic (ESD) measurements were used to calculate fractional pump flow (FPF), the product of contractile frequency (FREQ) measured as contractions per minute (Hz), and ejection fraction (EF) calculated as (EDD2-ESD2)/EDD2, at incremental intraluminal pressures ranging from 2 to 10 cmH2O. Statistical differences were determined by two-way ANOVA followed by Sidak correction for multiple comparisons. All data are means ± SEM. In sCLV isolated from 5 months-old males and females C57bl/6J mice, acute Aβ exposure significantly decreased FREQ (Vehicle vs Aβ, n=4 / n=7, p<0.01), without changes in EF (Vehicle vs Aβ, n=4 / n=7, p=0.115) or FPF (Vehicle vs Aβ, n=4 / n=7, p= 0.536). In pressurized sCLV isolated from 5x-FAD mice, when compared to wild-type littermates, we observed a significant reduction in FREQ (Wild-type vs 5x-FAD, n=4 / n=5, p<0.01). No differences were observed in EF (Wild-type vs 5x-FAD, n=4 / n=5, p=0.1224) or FPF (Wild-type vs 5x-FAD, n=4 / n=5, p=0.193). Together, these preliminary data suggest that acute exposure to Aβ(1-40) decreases FREQ of isolated sCLV, a similar effect observed in sCLV isolated from 5x-FAD mice, albeit not reaching statistical power for multiple comparison significance. Consequently, a decrease contractile frequency can contribute to impairments in lymphatic pumping, which to some extent, can be compensated by prolonged filling times, thus leading to increased ejection fraction. In conclusion, we predict that in vivo sCLV contractile function is maintained via compensatory increases in ejection fraction in conditions of exacerbated Aβ production and deposition. Funding: National Institutes of Health (R01 AG073230) and the Alzheimer’s Association (AARGD-21-850835). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Increased contractile activity and dilation of popliteal lymphatic vessels in the TNF-α-overexpressing TNFΔARE/+ arthritic mouse

AimsTNF-α acute treatment has been found to disrupt lymphatic drainage in the setting of arthritis through the NF-kB-iNOS- signaling pathway. We examined whether popliteal lymphatic vessels (pLVs) contractile activity was altered in 12- and 24- week-old females of an arthritic mouse model overexpressing TNF-α (TNFΔARE/+). Main methodspLVs were prepared for intravital imaging to measure lymph flow speed, and ex vivo functional responses to a stepwise increase in transmural pressure in the absence or presence of the non-selective NOS inhibitor (L-NNA) or the selective iNOS inhibitor (1400W) were compared between TNFΔARE/+ and WT mice. Total eNOS (t-eNOS) and eNOS phosphorylated at ser1177 (p-eNOS) were evaluated by western blotting. Key findingsIn vivo imaging revealed a significantly increase in lymph flow speed in TNFΔARE/+ mice in comparison to WT at both ages. Pressure myography showed an increase in contraction frequency, diameters and fractional pump flow at both ages, whereas amplitude and ejection fraction were significantly decreased in older TNFΔARE/+ mice. Additionally, contraction frequency was increased in the presence of 1400W, and systolic diameter was abolished with L-NNA in TNFΔARE/+ mice compared to WT. Significant increases in p-eNOS expression and neutrophil recruitment (MPO activity) were observed in TNFΔARE/+ mice compared to WT. SignificanceOur data reveal functional changes in pLVs, especially in advanced stage of arthritis. These alterations may be related to eNOS and iNOS response, which can affect drainage of the inflammatory content from the joints.

Impact of Goreisan components on rat mesenteric collecting lymphatic vessel pumping.

Goreisan is a traditional herbal formulation with diuretic properties tested as a clinical therapeutic to alleviate lymphedema in Japan. The present study aimed to determine how Goreisan and its five different components affect lymphatic pump function. Mesenteric collecting lymphatics were isolated from anesthetized Sprague-Dawley rats and mounted on resistance-matched glass micropipettes in a 37°C physiological salt solution bath for studies. Diameter was continuously measured to obtain the following lymphatic pump parameters: contraction frequency (CF), end diastolic diameter (EDD), and end systolic diameter (ESD), contraction amplitude (AMP), ejection fraction (EF), and fractional pump flow (FPF). Goreisan and each of its components (Cinnamomi Cortex, Atractylodis Rhizoma, Alismatis Rhizoma, Polyporus, and Poria) were applied to the bath at concentrations of 1-30 μg/mL. The results show that while Goreisan causes no significant changes to lymphatic pumping, Alismatis Rhizoma and Polyporus each significantly reduce CF and FPF. In addition, rats that received oral administration of Goreisan and Alismatis Rhizoma for 1 week had elevated expression of VEGFR-3 in their mesenteric collecting lymphatics. Collectively, the results suggest that some components of Goreisan have a direct, rapid impact on lymphatic pumping. These findings provide new insights but also raise new questions about the therapeutic potential of Goreisan in patients with secondary lymphedema.

Ex vivo Demonstration of Functional Deficiencies in Popliteal Lymphatic Vessels From TNF-Transgenic Mice With Inflammatory Arthritis.

Background: Recent studies demonstrated lymphangiogenesis and expansion of draining lymph nodes during chronic inflammatory arthritis, and lymphatic dysfunction associated with collapse of draining lymph nodes in rheumatoid arthritis (RA) patients and TNF-transgenic (TNF-Tg) mice experiencing arthritic flare. As the intrinsic differences between lymphatic vessels afferent to healthy, expanding, and collapsed draining lymph nodes are unknown, we characterized the ex vivo behavior of popliteal lymphatic vessels (PLVs) from WT and TNF-Tg mice. We also interrogated the mechanisms of lymphatic dysfunction through inhibition of nitric oxide synthase (NOS).Methods: Popliteal lymph nodes (PLNs) in TNF-Tg mice were phenotyped as Expanding or Collapsed by in vivo ultrasound and age-matched to WT littermate controls. The PLVs were harvested and cannulated for ex vivo functional analysis over a relatively wide range of hydrostatic pressures (0.5–10 cmH2O) to quantify the end diastolic diameter (EDD), tone, amplitude (AMP), ejection fraction (EF), contraction frequency (FREQ), and fractional pump flow (FPF) with or without NOS inhibitors Data were analyzed using repeated measures two-way ANOVA with Bonferroni’s post hoc test.Results: Real time videos of the cannulated PLVs demonstrated the predicted phenotypes of robust vs. weak contractions of the WT vs. TNF-Tg PLV, respectively. Quantitative analyses confirmed that TNF-Tg PLVs had significantly decreased AMP, EF, and FPF vs. WT (p < 0.05). EF and FPF were recovered by NOS inhibition, while the reduction in AMP was NOS independent. No differences in EDD, tone, or FREQ were observed between WT and TNF-Tg PLVs, nor between Expanding vs. Collapsed PLVs.Conclusion: These findings support the concept that chronic inflammatory arthritis leads to NOS dependent and independent draining lymphatic vessel dysfunction that exacerbates disease, and may trigger arthritic flare due to decreased egress of inflammatory cells and soluble factors from affected joints.

Axial stretch regulates rat tail collecting lymphatic vessel contractions

Lymphatic contractions play a fundamental role in maintaining tissue and organ homeostasis. The lymphatic system relies on orchestrated contraction of collecting lymphatic vessels, via lymphatic muscle cells and one-way valves, to transport lymph from the interstitial space back to the great veins, against an adverse pressure gradient. Circumferential stretch is known to regulate contractile function in collecting lymphatic vessels; however, less is known about the role of axial stretch in regulating contraction. It is likely that collecting lymphatic vessels are under axial strain in vivo and that the opening and closing of lymphatic valves leads to significant changes in axial strain throughout the pumping cycle. The purpose of this paper is to quantify the responsiveness of lympatic pumping to altered axial stretch. In situ measurements suggest that rat tail collecting lymphatic vessels are under an axial stretch of ~1.24 under normal physiological loads. Ex vivo experiments on isolated rat tail collecting lymphatics showed that the contractile metrics such as contractile amplitude, frequency, ejection fraction, and fractional pump flow are sensitive to axial stretch. Multiphoton microscopy showed that the predominant orientation of collagen fibers is in the axial direction, while lymphatic muscle cell nuclei and actin fibers are oriented in both circumferential and longitudinal directions, suggesting an axial component to contraction. Taken together, these results demonstrate the significance of axial stretch in lymphatic contractile function, suggest that axial stretch may play an important role in regulating lymph transport, and demonstrate that changes in axial strains could be an important factor in disease progression.

T-type, but not L-type, voltage-gated calcium channels are dispensable for lymphatic pacemaking and spontaneous contractions

The spontaneous contractions of collecting lymphatic vessels provide an essential propulsive force to return lymph centrally. These contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. In previous studies, T-type voltage-gated Ca2+ channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni2+. Our goal was to test this idea in a more definitive way using genetic knock out mice. First, we demonstrated through both PCR and immunostaining that mouse lymphatic muscle cells expressed Cav3.1 and Cav3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each T-type VGCC isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied ex vivo. Further, both WT and Cav3.1−/−; 3.2−/− double knock-out lymphatic vessels responded similarly to mibefradil and Ni2+, which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than T-type VGCCs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and Cav3.1−/−; 3.2−/− double knock-out mice. In contrast, smooth-muscle specific deletion of the L-type VGCC, Cav1.2, completely abolished all lymphatic spontaneous contractions. Collectively our results suggest that, although T-type VGCCs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and Ni2+ in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions.

Entrainment of Lymphatic Contraction to Oscillatory Flow

Lymphedema, a disfiguring condition characterized by an asymmetrical swelling of the limbs, is suspected to be caused by dysfunctions in the lymphatic system. A possible source of lymphatic dysfunction is the reduced mechanosensitivity of lymphangions, the spontaneously contracting units of the lymphatic system. In this study, the entrainment of lymphangions to an oscillatory wall shear stress (OWSS) is characterized in rat thoracic ducts in relation to their shear sensitivity. The critical shear stress above which the thoracic ducts show a substantial inhibition of contraction was found to be significantly negatively correlated to the diameter of the lymphangion. The entrainment of the lymphangion to an applied OWSS was found to be significantly dependent on the difference between the applied frequency and the intrinsic frequency of contraction of the lymphangion. The strength of the entrainment was also positively correlated to the applied shear stress when the applied shear was less than the critical shear stress of the vessel. The ejection fraction and fractional pump flow were also affected by the difference between the frequency of the applied OWSS and the vessel's intrinsic contraction frequency. The results suggest an adaptation of the lymphangion contractility to the existing oscillatory shear stress as a function of its intrinsic contractility and shear sensitivity. These adaptations might be crucial to ensure synchronized contraction of lymphangions through mechanosensitive means and might help explain the lymphatic dysfunctions that result from impaired mechanosensitivity.

Evaluation of the Impact of the Traditional Medicine Formulation Goreisan and Its Components on Mesenteric Lymphatic Vessel Contraction and Lymphatic Endothelial Barrier Function

Kampo medicines are traditional herbal formulas used to treat and prevent various diseases. The Kampo medicine Goreisan is a known diuretic and is currently being tested in the clinic to reduce symptoms of surgically induced lymphedema in OB/GYN patients in Japan. The present study was aimed to clarify how Goreisan and its five different components affect lymphatic pump function. Mesenteric collecting lymphatics were isolated from anesthetized male Sprague Dawley rats, and mounted on resistance‐matched glass micropipettes in a 37 °C physiological salt solution bath for studies. The luminal pressure was set at 2 cm H2O and vessels that established intrinsic phasic contractions were studied. Contraction frequency (CF), end diastolic diameter (EDD), and end systolic diameter (ESD) were measured. From these, amplitude (AMP), ejection fraction (EF), and fractional pump flow (FPF) were calculated. Goreisan was applied at concentrations of 1–30 μg/ml. Its components, Cinnamomi Cortex, Atractilodys Rhizoma, Alismatis Rhizoma, Polyporus, and Poria were also studied at concentrations of 1–30 μg/ml. To measure barrier function, human dermal lymphatic endothelial cells (LEC) were treated with 1–100 μg/ml of each compound and transendothelial electrical resistance (TER) was determined by electric cell‐substrate impedance sensing (ECIS). The results show that treatment with Goreisan causes no significant changes in all parameters compared to baseline. However, 30ug/ml Alismatis Rhizoma caused a significant reduction in CF and FPF. The other components did not affect pumping. Goreisan also does not significant affect TER. Polyporus significantly increased TER compared to baseline, while the other components elicited no change. Collectively, the results suggest that Goreisan does not directly affect lymphatic pumping or barrier function, although at its component Alismatis Rhizoma can influence pump activity and Polyporus can enhance barrier function. By extension, in the lymphedema patients treated with Goreisan, its pharmacological action is more likely to be on renal mechanism to reduce excess body fluids. Additional work is needed to determine whether Goreisan may have direct effect to rescue impaired lymphatic vessel functions under inflammatory conditions.Support or Funding InformationThis work was supported by Rotary International Global Grants Scholarship Grant Number GG1746618.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Temporal Dynamics of the Rat Thoracic Duct Contractility in the Presence of Imposed Flow.

The initial periods of increased flow inside lymphatic vessels demonstrate specific temporary patterns of self-tuning of lymphatic vessel contractility that are heterogeneous across regional lymphatic networks. The current literature primarily refers to the immediate and fast reactions of the lymphangions to increases in basal flow. Until now, there were no available data on how the lymphatic vessels react to comparatively longer periods of imposed flow. In this study, we measured and analyzed the contractility of the rat thoracic duct segments, isolated, cannulated, and pressurized at 3 cm H2O at no imposed flow conditions and during 4 hours of imposed flow (constant transaxial pressure gradient of 2 cm H2O). We found the development of a progressing lymphatic tonic relaxation and inhibition of the lymphatic contraction frequency over 4 hours of imposed flow. After a short initial decrease, lymphatic phasic contraction amplitude rose significantly during the first hour of imposed flow, and it demonstrated a trend to return toward control levels after 3 hours of imposed flow. As a result, the fractional pump flow (active lymph pumping per minute) of isolated thoracic duct segments reached and maintained a statistically significant decrease (from control no-flow conditions) at the end of the third hour of imposed flow. Our new findings provide a better understanding of how lymphatic contractility changes during the development of prolonged periods of steady lymph flow. The latter may occur during the initial phases of development of an inflammatory-related tissue edema.

Modulation of mesenteric collecting lymphatic contractions by σ1-receptor activation and nitric oxide production.

Recently, it has been reported that a σ-receptor antagonist could reduce inflammation-induced edema. Lymphatic vessels play an essential role in removing excess interstitial fluid. We tested the hypothesis that activation of σ-receptors would reduce or weaken collecting lymphatic contractions. We used isolated, cannulated rat mesenteric collecting lymphatic vessels to study contractions in response to the σ-receptor agonist afobazole in the absence and presence of different σ-receptor antagonists. We used RT-PCR and Western blot analysis to investigate whether these vessels express the σ1-receptor and immunofluorescence confocal microscopy to examine localization of the σ1-receptor in the collecting lymphatic wall. Using N-nitro-l-arginine methyl ester (l-NAME) pretreatment before afobazole in isolated lymphatics, we tested the role of nitric oxide (NO) signaling. Finally, we used 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence as an indicator to test whether afobazole increases NO release in cultured lymphatic endothelial cells. Our results show that afobazole (50-150 µM) elevated end-systolic diameter and generally reduced pump efficiency and that this response could be partially blocked by the σ1-receptor antagonists BD 1047 and BD 1063 but not by the σ2-receptor antagonist SM-21. σ1-Receptor mRNA and protein were detected in lysates from isolated rat mesenteric collecting lymphatics. Confocal images with anti-σ1-receptor antibody labeling suggested localization in the lymphatic endothelium. Blockade of NO synthases with l-NAME inhibited the effects of afobazole. Finally, afobazole elicited increases in NO production from cultured lymphatic endothelial cells. Our findings suggest that the σ1-receptor limits collecting lymphatic pumping through a NO-dependent mechanism.NEW & NOTEWORTHY Relatively little is known about the mechanisms that govern contractions of lymphatic vessels. σ1-Receptor activation has been shown to reduce the fractional pump flow of isolated rat mesenteric collecting lymphatics. The σ1-receptor was localized mainly in the endothelium, and blockade of nitric oxide synthase inhibited the effects of afobazole.

Reduction of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation.

To evaluate the changes of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation. Six rats in the shock group suffered hypotension for 90 min by hemorrhage, and resuscitation with shed blood and equal ringer's solution. Then, the contractility of lymphatics, obtained from thoracic ducts in rats of the shock and sham groups, were evaluated with an isolated lymphatic perfusion system using the indices of contractile frequency (CF), tonic index (TI), contractile amplitude (CA) and fractional pump flow (FPF). The lymphatic reactivity to substance P (SP) was evaluated with the different volume of CF, CA, TI and FPF between pre- and post-treatment of SP at different concentrations. The CF, FPF, and TI of lymphatics obtained from the shocked rats were significantly decreased than that of the sham group. After SP stimulation, the ∆CF (1 × 10(-8), 3 × 10(-8), 1 × 10(-7), 3 × 10(-7) mol/L), ∆FPF (1 × 10(-8), 3 × 10(-8), 1 × 10(-7) mol/L), and ∆TI (1 × 10(-8) mol/L) of lymphatics in the shock group were also obviously lower compared with the sham group. In addition, there were no statistical differences in CA and ∆CA between two groups. Lymphatic contractility and reactivity to substance P appears reduction following hemorrhagic shock with resuscitation.

Lymph Transport in Rat Mesenteric Lymphatics Experiencing Edemagenic Stress

To assess lymphatic flow adaptations to edema, we evaluated lymph transport function in rat mesenteric lymphatics under normal and increased fluid volume (edemagenic) conditions in situ. Twelve rats were infused with saline (intravenous infusion, 0.2 mL/min/100 g body weight) to induce edema. We intravitally measured mesenteric lymphatic diameter and contraction frequency, as well as lymphocyte velocity and density before, during, and after infusion. A 10-fold increase in lymphocyte velocity (0.1-1 mm/s) and a sixfold increase in flow rate (0.1-0.6 μL/min), were observed post infusion, respectively. There were also increases in contraction frequency and fractional pump flow one minute post infusion. Time-averaged wall shear stress increased 10 fold post infusion to nearly 1.5 dynes/cm(2) . Similarly, maximum shear stress rose from 5 to 40 dynes/cm(2) . Lymphatic vessels adapted to edemagenic stress by increasing lymph transport. Specifically, the increases in lymphatic contraction frequency, lymphocyte velocity, and shear stress were significant. Lymph pumping increased post infusion, though changes in lymphatic diameter were not statistically significant. These results indicate that edemagenic conditions stimulate lymph transport via increases in lymphatic contraction frequency, lymphocyte velocity, and flow. These changes, consequently, resulted in large increases in wall shear stress, which could then activate NO pathways and modulate lymphatic transport function.

Functional adaptation of bovine mesenteric lymphatic vessels to mesenteric venous hypertension

Lymph flow is the primary mechanism for returning interstitial fluid to the blood circulation. Currently, the adaptive response of lymphatic vessels to mesenteric venous hypertension is not known. This study sought to determine the functional responses of postnodal mesenteric lymphatic vessels. We surgically occluded bovine mesenteric veins to create mesenteric venous hypertension to elevate mesenteric lymph flow. Three days after surgery, postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 7) and sham surgery (Sham; n = 6) group animals were evaluated and compared. Contraction frequency (MVH: 2.98 ± 0.75 min(-1); Sham: 5.42 ± 0.81 min(-1)) and fractional pump flow (MVH: 1.14 ± 0.30 min(-1); Sham: 2.39 ± 0.32 min(-1)) were significantly lower in the venous occlusion group. These results indicate that postnodal mesenteric lymphatic vessels adapt to mesenteric venous hypertension by reducing intrinsic contractile activity.

Role of RhoA in Regulating the Pump Function of Isolated Lymphatics From Hemorrhagic Shock Rats

The aim of this present study was to examine changes in RhoA protein levels and the role in RhoA in lymphatic contractility and reactivity after hemorrhagic shock. Levels of RhoA and phospho-RhoA in lymphatic tissue isolated from hemorrhagic shock rats were measured, and the contractility and reactivity to substance P of lymphatics isolated from control rats and rats subjected to shock 0.5 and 2 h were determined with an isolated lymphatic perfusion system at a transmural pressure of 3 cmH2O. At the same time, lymphatics isolated from rats subjected to shock 0.5 and 2 h were incubated with agonists and antagonists of RhoA/Rho kinase signaling. Contractile frequency, end-diastolic and end-systolic diameter, and passive diameter were recorded and used to calculate lymphatic tonic index, contractile amplitude, and fractional pump flow. After stimulation with a gradient of substance P, the differences between the preadministration and postadministration values of contractile frequency, contractile amplitude, tonic index, and fractional pump flow were calculated to further assess lymphatic reactivity. RhoA protein levels were significantly increased at 0.5 h after shock but decreased at 2 and 3 h after shock; p-Rho levels were initially increased after shock and subsequently decreased. The contractility and reactivity of 0.5-h-shocked lymphatics were significantly reduced by the RhoA antagonist C3 transferase and the Rho kinase antagonist Y-27632. The RhoA agonist U-46619 increased the contractility and reactivity of 2-h-shocked lymphatics, whereas Y-27632 suppressed the effect of U-46619. Okadaic acid, an inhibitor of myosin light-chain phosphatase, had no effect on the contractility of 2-h-shocked lymphatics, but improved lymphatic reactivity. These results suggest that RhoA is involved in the modulation of lymphatic pump function during hemorrhagic shock and that its effects may be mediated by Rho kinase and MLCP.

Genetic removal of basal nitric oxide enhances contractile activity in isolated murine collecting lymphatic vessels

The role of nitric oxide (NO) in regulating lymphatic contractile function and, consequently, lymph flow has been the subject of intense study. Despite this, the precise effects of NO on lymphatic contractile activity remain unclear. Recent hypotheses posit that basal levels of endogenous NO increase lymphatic contraction strength as a consequence of lowering frequency (i.e. positive lusitropy), whereas higher agonist-evoked concentrations of NO exert purely inhibitory effects on contractile function. We tested both hypotheses directly by isolating and cannulating collecting lymphatic vessels from genetically modified mice for ex vivo study. The effects of basal NO and agonist-evoked NO were evaluated, respectively, by exposing wild-type (WT), endothelial NO synthase (eNOS)(-/-) and inducible NO synthase (iNOS)(-/-) lymphatic vessels to controlled pressure steps followed by ACh doses. To compare with pharmacological inhibition of eNOS, we repeated both tests in the presence of l-NAME. Surprisingly, genetic removal of basal NO enhanced contraction amplitude significantly without increasing contraction frequency. Higher levels of NO production stimulated by ACh evoked dilation, decreased tone, slowed contraction frequency and reduced fractional pump flow. We conclude that basal NO specifically depresses contraction amplitude, and that greater NO production then inhibits all other aspects of contractile function. Further, this work demonstrates definitively that mouse collecting lymphatic vessels exhibit autonomous, large-amplitude contractions that respond to pressure similarly to collecting lymphatics of other mammalian species. At least in the peripheral lymphatic vasculature, NO production depresses contractile function, which influences lymph flow needed for fluid regulation, humoral immunity and cancer metastasis.

ATP-sensitive potassium channel involved in modulation of nitride oxide regulating contractile activity of isolated lymphatics from hemorrhagic shock rats

To investigate the mechanism of ATP-sensitive potassium channel (K(ATP)) on nitride oxide (NO) regulating contractile activity of isolated lymphatics from hemorrhagic shock (HS) rats. Eighty - four Wistar rats were randomly divided into control group (n=6), HS 0.5-hour group (n=36), HS 2-hour group (n=42). A segment of thoracic duct of rats was adopted for isolated lymphatics after HS, then the HS 0.5-hour and 2-hour lymphatics were incubated combined or respectively with K(ATP) inhibitor glibenclamide (Gli), opener of K(ATP) pinacidil (Pin), NO donor L-arginine (L-Arg), protein kinase A (PKA) inhibitor H-89, PKA donor 8-bromine-cyclic adenosine monophosphate (8-Br-cAMP), nitricoxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME), soluble guanylyl cyclase (sGC) inhibitor 1 h-[1,2,4]-oxadiazole-[4,3-a]- quinoxalin-1-one (ODQ), protein kinase G (PKG) inhibitor KT-5823 (named as HS 0.5 h, HS 0.5 h + L-Arg, HS 0.5 h + L-Arg + H-89, HS 0.5 h + L-Arg + Gli, HS 0.5 h + 8-Br-cAMP, HS 0.5 h + 8-Br-cAMP + Gli and HS 2 h, HS 2 h + L-NAME, HS 2 h + L-NAME + Pin, HS 2 h + ODQ, HS 2 h + ODQ + Pin, HS 2 h + KT-5823, HS 2 h + KT-5823 + Pin, n=6). By lymphatic perfusion in vitro, contractile frequency (CF) was recorded, and contractile amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were calculated. The results suggested that the CF, TI, FPF of HS 0.5-hour lymphatics were significantly increased compared with control group, and the CF, TI, FPF decreased significantly when incubated with L-Arg. H-89 could deteriorate the decreased effect of L-Arg on CF and FPF, and Gli could deteriorate the decreased effect of L-Arg on FPF. When the HS 0.5-hour lymphatics incubated with 8-Br-cAMP, the CF and FPF were all decreased significantly, and when the HS 0.5-hour lymphatics incubated with 8-Br-cAMP and Gli, the CF was significantly higher than HS 0.5 h + 8-Br-cAMP group, and the TI and FPF decreased significantly compared with HS 0.5-hour group. The CF, FPF, TI of HS 2-hour lymphatics were significantly decreased compared with control group. L-NAME could increase the CF, TI, FPF; ODQ could increase the CF, TI; KT-5823 could increase the CF and FPF; when incubated with Pin respectively, the CF and FPF when incubated with L-NAME were decreased, the CF, TI and FPF when incubated with ODQ were decreased, in addition, the CA was recovered as level of control group, the CF and FPF when incubated with KT-5823 were decreased. K(ATP) involved in NO modulating pumping function of isolated lymphatics of HS rats, and the effect may be relative to the signal pathway of NO-cAMP-PKA and NO-cGMP-PKG.

Aging-Associated Alterations in Contractility of Rat Mesenteric Lymphatic Vessels

To evaluate the age-related changes in pumping of mesenteric lymphatic vessels in 9- and 24-month-old male Fisher-344 rats. Lymphatic diameters, contraction amplitude, contraction frequency, and fractional pump flow were determined in isolated MLV before and after l-NAME application. The data demonstrate a severe weakening of the lymphatic pump in aged MLV including diminished lymphatic contraction amplitude, contraction frequency, and as a result, lymphatic pump activity. The data also suggest that the imposed flow gradient-generated shear-dependent relaxation does not exist in aged rat MLV, and the sensitivity of both adult and aged MLV to such shear cannot be eliminated by nitric oxide (NO) synthases blockade. These data provide new evidence of lymphatic regional heterogeneity for both adult and aged MLV. In MLV, a constant interplay between the tonic and phasic components of the myogenic response and the shear-dependent release of NO predominantly determine the level of contractile activity; the existence of another shear-dependent, but NO-independent regulatory mechanism is probably present. Aging remarkably weakens MLV contractility, which would predispose this lymphatic network to lower total lymph flow in resting conditions and limit the ability to respond to an edemagenic challenge in the elderly.

Age‐Related Alterations of Active Pumping Mechanisms in Rat Thoracic Duct

To evaluate the age-related changes in active pumping in thoracic duct (TD) from 24-month-old Fisher-344 rats comparing with TD pumping in 9-month rats. Lymphatic diameters, contraction amplitude and frequency, ejection fraction, and fractional pump flow were determined in isolated TD preparations. Western blot analyses were performed to evaluate relative levels of eNOS and iNOS in 9- and 24-month-old TD. Stretch-dependent regulation was altered in aged TD especially at higher levels of pressure: the negative inotropy, negative chronotropy and diminished minute pumping (2- to 3-fold decrease) were observed. Physiological NO/imposed-flow-dependent inhibition was completely abolished in aged TD, yet NO-synthase blockade by L-NAME (10(-4) M) increased pumping in a flow-independent manner. Western blot analyses indicated that the relative levels of eNOS were decreased approximately 7-fold in the 24-month-old TD when compared with 9-month-old TD; whereas iNOS levels were increased approximately 10-fold in 24-month-old TD. These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function.