Lymphatic Contractile Function Research Articles

Not just fibrotic: endothelial-derived TGFβ maintains contractile function and lymphatic muscle phenotype during homeostasis.

Cell-cell communication within the lymphatic vasculature during homeostasis is incompletely detailed. Although many discoveries highlight the pathological roles of transforming growth factor-beta (TGFβ) in chronic vascular inflammation and associated fibrosis, only a small amount is known surrounding the role of TGFβ-signaling in homeostatic lymphatic function. Here, we discovered that pharmacological blockade of TGFβ receptor 1 (TGFβR1) negatively impacts rat mesenteric lymphatic vessel pumping, significantly reducing vessel contractility and surrounding lymphatic muscle coverage. We have identified mesenteric lymphatic endothelial cells themselves as a source of endogenous vascular TGFβ and that TGFβ production is significantly increased in these cells via activation of a number of functional pattern recognition receptors they express. We show that a continuous supply of TGFβ is essential to maintain the contractile phenotype of neighboring lymphatic muscle cells and support this conclusion through in vitro analysis of primary isolated lymphatic muscle cells that undergo synthetic differentiation during 2-D cell culture, a phenomenon that could be effectively rescued by supplementation with recombinant TGFβ. Finally, we demonstrate that lymphatic endothelial production of TGFβ is regulated, in part, by nitric oxide in a manner we propose is essential to counteract the pathological over-production of TGFβ. Taken together, these data highlight the essential role of homeostatic TGFβ signaling in the maintenance of lymphatic vascular function and highlight possible deleterious consequences of its inhibition.NEW & NOTEWORTHY The growth factor TGFβ is commonly associated with its pathological overproduction during tissue fibrosis rather than its homeostatic functions. We expose the lymphatic endothelium as a source of endogenous TGFβ, the impact of its production on the maintenance of surrounding lymphatic muscle cell phenotype, and internally regulated mechanisms of its production. Overall, these results highlight the intricate balance of TGFβ-signaling as an essential component of maintaining lymphatic contractile function.

Role of TRPV4 in superficial cervical lymphatic vessel contractile function

Collecting cervical lymphatic vessels pump lymph and macromolecules drained by brain meningeal lymphatics to the cervical lymph nodes. Collecting lymphatics vessels rely on spontaneous contractions to propel lymph against a pressure gradient to re-enter the venous circulation. The calcium permeable transient receptor potential vanilloid 4 channel (TRPV4) has been shown to mediate intraluminal pressure-induced vasodilatory responses in small arteries. However, whether TRPV4 participates in pressure-dependent contractile responses in lymphatic vessels remains unclear. We hypothesized that increases in intraluminal pressure leads to TRPV4-dependent increases in lymphatic pump function. TRPV4 expression was assessed by PCR assay in isolated superficial cervical lymphatic vessels (sCLV). Using pressure myography, contractile function of isolated sCLV was assessed in the presence of TRPV4 antagonist (1μM HC-067047) or vehicle (DMSO). To quantify lymphatic pump function (FPF), the product of contractile frequency (FREQ) and ejection fraction (EF) was analyzed using repeated end-diastolic and end-systolic diameter measurements. We detected TRPV4 mRNA expression following PCR amplification from isolated sCLV tissue of young C57Bl/6J mice (4-6 months). In pressurized sCLV, TRPV4 activation with GSK 1016790A (10 nM) abolished spontaneous rhythm contractions by causing a sustained constriction, consequently leading to a significant decrease in lymphatic pump function (FPF: 4.67±0.58 vs 1.17±0.86, Vehicle vs GSK 1016790A, n=8, p<0.05) at baseline intraluminal pressure (3 cm H2O). TRPV4 antagonism (HC-067047, 1 μM) did not affect basal sCLV contractility (FPF: 5.15±0.6 vs 3.44±0.43, Vehicle vs HC 067047, n=5, p>0.05). Further, when HC-067047 was applied to the bath and intraluminal pressure was maintained at 2 cm H2O, no changes were observed in FREQ (9.3±1.4 vs 11.7±1.3 Hz, Vehicle vs. HC-067047, n=7 / 9, p>0.05), EF (0.253±0.040 vs 0.268±0.037, Vehicle vs. HC-067047, n=7 / 9, p>0.05), or contractile amplitude (22.54±6.769 vs 26.76±3.028 μm, Vehicle vs HC-067047, n=7 / 9, p>0.05). Similarly, TRPV4 blockade did not alter FREQ (12.4±1.3 vs 14.3±1.4 Hz, Vehicle vs. HC-067047, n=7 / 9, p>0.05), EF (0.090±0.024 vs 0.092±0.019, Vehicle vs HC-067047) or contractile amplitude (7.98±2.814 vehicle vs 9.27±1.88 μm, Vehicle vs HC-067047) at the higher intraluminal pressure of 7 cm H2O. Collectively, these data suggest that TRPV4 channels are not involved in pressure-dependent contractile responses of sCLV in mice, despite inducing a robust constriction upon pharmacological activation. 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 2023 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.

A vascular smooth muscle-specific integrin-α8 Cre mouse for lymphatic contraction studies that allows male-female comparisons and avoids visceral myopathy.

Introduction: The widely-used, tamoxifen-inducible, smooth muscle (SM)-specific Cre, Myh11-CreERT2 , suffers from two disadvantages: 1) it is carried on the Y-chromosome and thus only effective for gene deletion in male mice, and 2) it recombines in both vascular and non-vascular SM, potentially leading to unwanted or confounding gastrointestinal phenotypes. Here, we tested the effectiveness of a new, SM-specific Cre, based on the integrin α8 promoter (Itga8-CreERT2 ), that has been recently developed and characterized, to assess the effects of Cav1.2 deletion on mouse lymphatic SM function. Methods: Cav1.2 (the L-type voltage-gated calcium channel) is essential for lymphatic pacemaking and contraction and its deletion using either Myh11-CreERT2 or Itga8-CreERT2 abolished spontaneous lymphatic contractions. Mouse lymphatic contractile function was assessed using two ex vivo methods. Results: Myh11-CreERT2 ; Cav1.2 f/f mice died of gastrointestinal obstruction within 20 days of the first tamoxifen injection, preceded by several days of progressively poor health, with symptoms including weight loss, poor grooming, hunched posture, and reduced overall activity. In contrast, Itga8-CreERT2 ; Cav1.2 f/f mice survived for >80days after induction and were in normal health until the time of sacrifice for experimental studies. Cav1.2 deletion was equally effective in male and female mice. Discussion: Our results demonstrate that Itga8-CreER T2 can be used to effectively delete genes in lymphatic smooth muscle while avoiding potentially lethal visceral myopathy and allowing comparative studies of lymphatic contractile function in both male and female mice.

Evaluation of Longitudinal Lymphatic Function Changes upon Injury in the Mouse Tail with Photodynamic Therapy.

The lymphatic system is an essential but often understudied component of the circulatory system in comparison with its cardiovascular counterpart. Such disparity could often be explained by the difficulty in imaging lymphatics and the specialized microsurgical skills that are often required for lymphatic injury models. Recently, it has been shown that verteporfin, a photosensitive drug used for photodynamic therapy (PDT) to ablate the blood vessels, provides a similar effect on lymphatic vessels. Here, we seek to administer verteporfin and perform a modified form of PDT on collecting lymphatics in the mouse tail, a commonly used location for the study of lymphatic disorders, and examine lymphatic remodeling, contractility, and transport in response to the procedure. Mice collecting lymphatics in the tail were injured by PDT through an intradermal injection of verteporfin in the distal tip of the tail followed by light activation on the proximal portion of the tail downstream of the injection site. Lymphatic function was evaluated using a near-infrared (NIR) imaging system weekly for up to 28days after injury. PDT resulted in a loss in lymphatic function contractile frequency that persisted for up to 7days after injury. Packet transport and packet amplitude, measurements reflective of the strength of contraction, were significantly reduced 14days after injury. The lymphatics showed a delayed increase in lymphatic leakage at 7days that persisted until the study endpoint on day 28. This technique provides an easy-to-use method for injuring lymphatics to understand their remodeling response to injury by PDT as well as potentially for screening therapeutics that seek to normalize lymphatic permeability or contractile function after injury.

Hypertension is linked to enhanced lymphatic contractile response via RGS16/RhoA/ROCK pathway.

Lymph capillary network can be expected to alter blood pressure via regulating interstitial electrolyte and volume balance. However, the pathophysiology of lymphatic vessel in hypertension is poorly understood. In this study, we examined lymph vessel function focusing on contractile response in hypertensive rats. It was found that thoracic ducts isolated from adult (10-14 wk old) spontaneously hypertensive rats (SHRs) exhibited increased agonist-mediated contraction compared with age-matched Wistar-Kyoto (WKY) rats, whereas lymphatic contractions in younger (4 wk old) SHRs, exhibiting normal blood pressure, were no different compared with age-matched control rats. Tight regulation of blood pressure with antihypertensive drugs (hydrochlorothiazide/hydralazine) did not prevent the augmented lymphatic contraction in adult SHRs; however, treatment of SHRs with angiotensin II (ANG II) type 1 receptor blocker (losartan) for 6 wk abolished the augmentation of lymphatic contractions. In addition, ANG II infusion in Wistar rat caused augmented lymphatic contractile responses in the thoracic duct. The augmented contractions in adult SHRs were diminished by a ROCK inhibitor (Y-27632). Consistently, the thoracic ducts in SHRs showed significantly higher phosphorylation of myosin phosphatase targeting protein-1 than WKY rats. Furthermore, gene expression profiling of adult SHR lymphatics showed marked loss of regulator of G-protein signaling 16 (RGS16) mRNA, which was confirmed by the real-time PCR. Treatment with the RGS inhibitor CCG-63808 enhanced contractions in thoracic ducts from Wistar rats, which were abolished by the ROCK inhibitor. It is concluded that lymphatic contractile function was enhanced in hypertensive model rats, which could be mediated by dysregulation of the ROCK pathway possibly through RGS16.NEW & NOTEWORTHY Lymph capillary controls interstitial electrolyte and volume balance, which may blunt increased blood pressure. However, the function of lymphatic vessel in hypertension is poorly understood. Our study showed that the lymphatic smooth muscle contractility is hyperreactive in two different hypertensive models. The lymphatic dysfunction could be mediated by dysregulation of ROCK pathway possibly through RGS16. The present finding supports a new concept showing the functional relationship between lymphatic contractile activity and hypertension.

Lymphatic Function Decreases over Time in the Arms of Breast Cancer Patients following Treatment

Background:In patients with breast cancer-related lymphedema, distinct lymphatic patterns and changed lymphatic contractile function have been described, but it is unknown how these characteristics change over time and to what extent they appear before clinical edema is detectable. Recently, we described the lymphatic morphology and function in a cohort of breast cancer patients shortly after radiation therapy (RT). In the current study, we investigate lymphatic function and morphology in the same cohort after 1 year of follow-up.Methods:The study population consisted of 28 breast cancer patients investigated 12 months after adjuvant locoregional RT. Lymphatic contraction frequency (CF), propulsion velocity, and the morphology of lymphatic vessels in the upper extremities were described in vivo using near-infrared fluorescence imaging. Lymphatic stress test was performed using hyperthermia.Results:At 1 year after RT, (n = 28) 46% of the patients presented with lymphatic morphological abnormalities with a degree of dermal backflow and 21% had developed clinical breast cancer-related lymphedema. In the ipsilateral arm, CF was 23% lower than in the contralateral arm (P = 0.04). Since primary examination, CF in the ipsilateral arm decreased by 40% (P = 0.03), whereas no change was observed in the contralateral arm. During hyperthermia, the ipsilateral arms with lymphatic complications were not able to increase CF as the remaining subgroups.Conclusions:Lymphatic function in the ipsilateral arm deteriorated over time after adjuvant breast cancer therapy. Furthermore, the presence of abnormal torturous lymphatic vessels in asymptomatic arms appeared to be associated with weak lymphatic reserve pumping capacity.

Vmeasur: A software package for experimental and clinical measurement of mesenteric lymphatic contractile function over an extended vessel length.

Conventionally, in vivo mesenteric lymphatic contractile function is measured using a high magnification transmission microscope (field of view 0.3-1.5mm), which precludes visualization of extended lengths of vessels embedded in mesenteric fat. Existing software is not optimized for imaging at a low magnification using a contrast agent. We aimed to develop a simple and clinically transferable method for in situ visualization, image analysis, and quantitative assessment of mesenteric lymphatic contractile function over an extended area. Subserosal injection of various blue dyes was taken up by mesenteric lymphatics and visualized under a stereomicroscope (25×), allowing for video recording of 1.4×1.1cm of intact mesentery. A new R package ("vmeasur") that combines multiple high-performance image analyses into a single workflow was developed. The edges of each vessel were determined by applying an automated threshold to each frame (with real-time manual verification). The vessel width at every point in each frame was plotted to provide contractile parameters over time and along the lymphatic vessel length. Contractile parameters and their differences along the length of the vessel were accurately calculated in a rodent model. In a human mesenteric lymphatic, the algorithm was also able to measure changes in diameter over length. This software offers a low cost, rapid, and accessible method to measure lymphatic contractile function over a wide area, showing differences in contractility along the length of a vessel. Because the presence of mesenteric fat has less of an impact on imaging, due to the use of an exogenous contrast agent, there is potential for clinical application.

Lymphatic contractile function: a comprehensive review of drug effects and potential clinical application.

The lymphatic system and the cardiovascular (CV) system work together to maintain body fluid homeostasis. Despite that, the lymphatic system has been relatively neglected as a potential drug target and a source of adverse effects from CV drugs. Like the heart, the lymphatic vessels undergo phasic contractions to promote lymph flow against a pressure gradient. Dysfunction or failure of the lymphatic pump results in fluid imbalance and tissue oedema. While this can be due to drug effects, it is also a feature of breast cancer-associated lymphoedema, chronic venous insufficiency, congestive heart failure, and acute systemic inflammation. There are currently no specific drug treatments for lymphatic pump dysfunction in clinical use despite the wealth of data from pre-clinical studies. The aim of this study was to identify (i) drugs with direct effects on lymphatic tonic and phasic contractions with potential for clinical application, and (ii) drugs in current clinical use that have a positive or negative side effect on lymphatic function. We comprehensively reviewed all studies that tested the direct effect of a drug on the contractile function of lymphatic vessels. Of the 208 drugs identified from 193 studies, about a quarter had only stimulatory effects on lymphatic tone, contraction frequency, and/or contraction amplitude. Of Food and Drug Administration-approved drugs, there were 14 that increased lymphatic phasic contractile function. The most frequently used class of drugs with inhibitory effects on lymphatic pump function were the calcium channels blockers. This review highlights the opportunity for specific drug treatments of lymphatic dysfunction in various disease states and for avoiding adverse drug effects on lymphatic contractile function.

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01–3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01–100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01–10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema.SIGNIFICANCE STATEMENTATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

Degradation of lymphatic anatomy and function in early venous insufficiency

ObjectiveWe used near-infrared fluorescence lymphatic imaging in a pilot study to assess the lymphatics in preulcerative (C2-C4) venous insufficiency and determine whether involvement and/or degradation of lymphatic anatomy or function could play a role in the progression of chronic venous insufficiency. We also explored the role of lymphatics in early peripheral arterial disease. MethodsAfter informed consent and intradermal injections of indocyanine green for rapid lymphatic uptake, near-infrared fluorescence lymphatic imaging was used to assess the lymphatic anatomic structure and quantify the lymphatic propulsion rates in subjects with early venous insufficiency. The anatomic observations included interstitial backflow, characterized by the abnormal spreading of indocyanine green from the injection site primarily into the surrounding interstitial tissues; dermal backflow, characterized by the retrograde movement of dye-laden lymph from collecting lymphatics into the lymphatic capillaries; and lymphatic vessel segmentation and dilation. ResultsTen subjects with venous insufficiency were enrolled, resulting in two legs with C2 disease, nine legs with C3 disease, eight legs with C4 disease, and one leg with C5 disease. Interstitial and/or dermal backflow were observed in 25%, 33%, and 41% of the injection sites in each limb with C2, C3, and C4 disease, respectively. Distinct vessel segmentation and dilation were observed in limbs with a C3 and higher classification, and dermal backflow proximal to the injection sites was observed in two legs with C4 disease and in the inguinal region of the C5 study subject. The overall average lymph propulsion rates were 1.3 ± 0.4, 1.2 ± 0.7, and 0.8 ± 0.5 contractile events/min for limbs with C2, C3, and C4 disease, respectively. One subject with peripheral arterial disease, who had previously undergone bypass surgery, presented with extensive dermal backflow and lymphatic reflux. ConclusionsNear-infrared fluorescence lymphatic imaging demonstrated that, compared with normal health subjects, the lymphatic anatomy and contractile function generally degrade with the severity of venous insufficiency. Lymphatic abnormalities mimic those in early cancer-acquired lymphedema subjects, as previously observed by us and others. Additional studies are needed to decipher the relationship, including any causality, between lymphatic dysfunction and peripheral vascular disease and venous insufficiency.

Abstract P079: Lymphatic Contraction Was Enhanced In Spontaneously Hypertensive Rats

Because lymph capillary network regulates interstitial electrolyte and volume balance, this system may function as regulating blood pressure (BP). Thus, changes in lymphatic activity can be expected to alter systemic BP. We recently reported that increasing BP caused lymphatic endothelial dysfunction via oxidative stress in hypertension. In this study, we examined lymphatic contractile function in thoracic duct from 10-14-week-old Wister-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). No distinct morphological difference was observed in thoracic duct isolated from WKY and SHR (n=4). Thoracic duct from SHR exhibited enhanced contractile response to serotonin (5-HT) or endothelin-1 (ET-1) compared to age-matched WKY (5-HT: 127.8±19.7% vs 53.6±10.3% of KCl-induced maximum contraction, p<0.05, n=5). The augmented contraction in SHR was diminished by fasudil, ROCK inhibitor (p<0.05, n=4-5). Consistent with this, increased phosphorylation of myosin phosphatase targeting protein (MYPT) was observed in SHR thoracic duct. Expression of ROCK1 or ROCK2 was not changed in SHR thoracic duct. Next we examined lymphatic contractile function in 4-week-old SHR showing normal BP. Contractile response to 5-HT or ET-1 was not enhanced in young SHR compared to age-matched WKY (n=4). To examine if lymphatic dysfunction was associated with increased BP, we treated 4-week-old SHR with antihypertensive drugs (hydrochlorothiazide and hydralazine) for 6 weeks. This treatment completely blocked elevation of systolic BP (SBP) in SHR (p<0.05, n=5), but did not prevent the augmentation of lymphatic contraction (5-HT: 99.0±7.2% vs 98.1±14.5%, n=5). Interestingly, treatment of young SHR with losartan (Angiotensin II type 1 receptor [AT1R] blocker) for 6 weeks ameliorated the augmented contraction (5-HT: 137.4±12.7% vs 77.9±8.8%, p<0.05, n=5), while this treatment scarcely diminished the increase in SBP (152.0±8.7 mmHg vs. 177.2±10.9 mmHg, p<0.05, n=5). We conclude that lymphatic contractile function was significantly enhanced in adult hypertensive SHR, which is mediated by RhoA/ROCK pathway thorough activation of AT1R. The increase in lymphatic constriction may be involved in increased BP in hypertension through decreased lymphatic compliance.

Kir6.1-dependent KATP channels in lymphatic smooth muscle and vessel dysfunction in mice with Kir6.1 gain-of-function.

Spontaneous contractions are essential for normal lymph transport and these contractions are exquisitely sensitive to the KATP channel activator pinacidil. KATP channel Kir6.1 and SUR2B subunits are expressed in mouse lymphatic smooth muscle (LSM) and form functional KATP channels as verified by electrophysiological techniques. Global deletion of Kir6.1 or SUR2 subunits results in severely impaired lymphatic contractile responses to pinacidil. Smooth muscle-specific expression of Kir6.1 gain-of-function mutant (GoF) subunits results in profound lymphatic contractile dysfunction and LSM hyperpolarization that is partially rescued by the KATP inhibitor glibenclamide. In contrast, lymphatic endothelial-specific expression of Kir6.1 GoF has essentially no effect on lymphatic contractile function. The high sensitivity of LSM to KATP channel GoF offers an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1 or SUR2, and suggests that glibenclamide may be an appropriate therapeutic agent. This study aimed to understand the functional expression of KATP channel subunits in distinct lymphatic cell types, and assess the consequences of altered KATP channel activity on lymphatic pump function. KATP channel subunits Kir6.1 and SUR2B were expressed in mouse lymphatic muscle by PCR, but only Kir6.1 was expressed in lymphatic endothelium. Spontaneous contractions of popliteal lymphatics from wild-type (WT) (C57BL/6J) mice, assessed by pressure myography, were very sensitive to inhibition by the SUR2-specific KATP channel activator pinacidil, which hyperpolarized both mouse and human lymphatic smooth muscle (LSM). In vessels from mice with deletion of Kir6.1 (Kir6.1-/- ) or SUR2 (SUR2[STOP]) subunits, contractile parameters were not significantly different from those of WT vessels, suggesting that basal KATP channel activity in LSM is not an essential component of the lymphatic pacemaker, and does not exert a strong influence over contractile strength. However, these vessels were >100-fold less sensitive than WT vessels to pinacidil. Smooth muscle-specific expression of a Kir6.1 gain-of-function (GoF) subunit resulted in severely impaired lymphatic contractions and hyperpolarized LSM. Membrane potential and contractile activity was partially restored by the KATP channel inhibitor glibenclamide. In contrast, lymphatic endothelium-specific expression of Kir6.1 GoF subunits had negligible effects on lymphatic contraction frequency or amplitude. Our results demonstrate a high sensitivity of lymphatic contractility to KATP channel activators through activation of Kir6.1/SUR2-dependent channels in LSM. In addition, they offer an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1/SUR2.

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.

Uncovering Endothelium‐Dependent Mechanisms of Lymphatic Dysfunction in Obesity

Obesity and type‐2 diabetes are major risk factors for the development of cardiovascular diseases. While evidence of lymphatic dysfunction has been reported in obese and/or diabetic patients, the mechanisms underlying that dysfunction are unknown. Efficient lymph transport relies on the intrinsic spontaneous contractions of collecting lymphatics and one‐way check valves that prevent or minimize backflow. Lymphatic contractions are initiated by action potentials that originate at a pacemaking site and then rapidly propagate between the strongly‐coupled lymphatic muscle cells (LMCs). We recently showed that in healthy lymphatics, electrical coupling between LMCs and lymphatic endothelial cells (LECs) appears to be quite limited, in contrast with strong coupling through myoendothelial junctions (MEJs) in arterioles. This apparent electrical isolation of LMCs from other surrounding cell networks may not only be a unique feature of the lymphatic vasculature, but it may be essential for the focal generation of pacemaking currents and their efficient propagation solely along the LMC‐layer. Therefore, we hypothesize that electrical coupling between LMCs and other cells can be abnormally enhanced in certain disease states that involve modifications to the extracellular matrix scaffold leading to abnormal cellular adhesion and abnormal cellular interactions. In particular, enhanced electrical coupling of LMCs and LECs could result in impaired lymphatic contractile function as a consequence of aberrant propagation of pacemaking signals. Lymphatic dysfunction, both contractile and valve dysfunction, could also result from increased fibrosis and stiffness in these disease states. Here we focus on the study of lymphatic dysfunction associated with obesity. Our results show that in obese mice (mice fed a Western Diet for 14 weeks), the contractile function of collecting lymphatics is impaired and lymphatic valve function decreased. While the origin for increased stiffening has not been determined, our preliminary observations point to lymphatics from obese mice being stiffer. Furthermore, PAI‐1 (Plasminogen Activator Inhibitor), a valuable biomarker in metabolic syndrome and type‐2 diabetes associated with obesity, is upregulated in the wall of lymphatic vessels from obese mice when compared to non‐obese age‐matched controls. Importantly, PAI‐1 has been shown to regulate the formation of vascular MEJs. Finally, our preliminary observations show that genetic overexpression of PAI‐1 in non‐obese mice results in apparently stiffer lymphatic vessels that display impaired contractility, while PAI‐1 deficiency appears to improve contractile function. In conclusion, our results associate obesity with lymphatic contractile dysfunction; point to a critical role for PAI‐1, which is primarily secreted by the endothelium, in regulating lymphatic function and regulating how cells interact in the lymphatic wall; and suggest the potential use of PAI‐1 as a therapeutic target to ameliorate lymphatic dysfunction associated with obesity.Support or Funding InformationThis research is supported by NIH‐1K99HL141143‐01A1 to JACG and NIH‐5R01HL125608‐02 to MJD.

SUBSTANCE P REGULATES INFLAMMATORY PATHWAYS IN LYMPHATIC MUSCLE

Substance P (SP), a neuropeptide that acts through the neurokinin receptors is associated with the sensory innervation of lymphoid tissue and modulates lymphatic contractile function and lymph flow. SP signaling is associated with several inflammatory pathologies. The role of lymphatics in body fluid homeostasis, lipid absorption and inflammation and immunity rely on the contractility of the lymphatic muscle cells (LMCs). We have shown that lymphatic contractile dysfunction is associated with perturbations in inflammatory mechanisms and that SP activates both inflammatory and contractile pathways in lymphatics. However, the specific inflammatory pathways and the resultant cytokines activated by SP in LMCs is unknown. We hypothesized that SP stimulates the pro‐survival AKT and MAPK pathways, and increases pro‐inflammatory cytokines. Cultured rat mesenteric LMCs were treated with SP (1 mM) and total RNA and protein were isolated at different time points (6 hr and 24 hr). Western analysis was carried out to detect the total and phosphorylated forms of AKT, JNK, GSK3b, b‐catenin, FOXO1 and PDCD4. b‐actin was used as control. The relative levels of inflammatory cytokines and chemokines, and specific inflammatory microRNAs (miRNAs) were analyzed by real time PCR. Our results indicate that SP activates the AKT and JNK pathways to significantly increase inflammatory cytokine and chemokine expression, and induces expression of miR 17–5p, miR19b and miR186 in the LMCs. Additionally, SP stimulates expression of genes, involved in lymphatic remodeling. Taken together our data demonstrate that SP activates multiple inflammatory signaling pathways in the LMCs that contribute to an inflammatory response in lymphatics during pathological conditions.Support or Funding InformationAmerican Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methods for Assessing the Contractile Function of Mouse Lymphatic Vessels Ex Vivo.

Lymphatic contractile dysfunction has been identified in several diseases, including lymphedema, yet a detailed molecular understanding of lymphatic muscle physiology has remained elusive. With the advent of genetic methods to manipulate gene expression in mice, a set of new tools became available for the investigation and visualization of the lymphatic vasculature. To gain insight into the molecular regulators of lymphatic contractile function, regulated primarily by the muscle cell layer encircling lymphatic collecting vessels, ex vivo approaches to allow control of hydrostatic and oncotic pressures and flow have been invaluable, complementing in vivo methods. While the original ex vivo techniques were developed for lymphatic vessels from large animals, and later adapted to rat vessels, here we describe modifications that enable the study of isolated, pressurized murine lymphatic collecting vessels. These methods, used in combination with transgenic mice, can be a powerful tool to investigate the molecular and cellular mechanisms of lymphatic function.

Fluorescence imaging of lymphatic outflow of cerebrospinal fluid in mice

Cerebrospinal fluid (CSF) is known to be reabsorbed by the lymphatic vessels and drain into the lymph nodes (LNs) through peripheral lymphatic vessels. In the peripheral lymphatics, the contractile pumping action of lymphangions mediates lymph drainage; yet it is unknown whether lymphatic vessels draining cranial and spinal CSF show similar function. Herein, we used non-invasive near-infrared fluorescence imaging (NIRFI) to image (i) indocyanine green (ICG) distribution along the neuraxis and (ii) routes of ICG-laden CSF outflow into the lymphatics following intrathecal lumbar administration. We demonstrate lymphatic contractile function in peripheral lymphatics draining from the nasal lymphatics to the mandibular LNs. In addition, we observed afferent sciatic lymphatic vessels, which also show contractile activity and transport spinal CSF into the sciatic LNs. This drainage pattern was also visualized by NIRFI following intrathecal thoracic injection. In situ intravital imaging following intrathecal lumbar injection of blue dye shows similar distributions to that seen in vivo with ICG. NIRFI could be used as a tool to probe CSF pathology including neurological disorders by imaging CSF outflow dynamics to lymphatics.

Abstract IA14: Lymphatic metastasis and the lymph node microenvironment

Abstract Background: Lymph nodes are the most common sites of solid tumor metastases. Their presence signals a poorer prognosis and the recommendation for systemic therapy in most cancer patients. However, why lymph node metastases are such strong predictors of outcome in cancer patients is the subject of much debate. Furthermore, tumor draining LNs commonly have an environment which suppresses immune response. Here we will explore the microenvironment of metastatic lymph nodes and tumor draining lymphatic vasculature to determine how cancer cells can spread and grow in the lymph node. Methods: We will measure lymphatic contractile function in tumor draining lymphatic vessels using intravital microscopy of an afferent lymphatic vessel to the popliteal lymph node (PLV). We will further use models that modify nitric oxide signaling to study how nitric oxide alters lymphatic pumping of tumor draining lymphatic vessels. Additionally, we will use our novel chronic lymph node window (CLNW) model to longitudinally image the growth and spread of lymph node metastases using intravital optical microscopy techniques. We use the CLNW in multiple models of spontaneous lymphatic metastases in mice to study the vasculature of metastatic lymph nodes. We further test our results in murine and patient samples using molecular and biochemical methods. Finally, we test the ability of antiangiogenic therapy to inhibit metastatic growth in the CLNW. Results: We show that lymphatic pumping is impaired in tumor draining lymphatic vessels and is mediated by nitric oxide signaling. We also show that lymph node metastases originate from multiple tumor cells not a single clonal progenitor. We reveal the surprising lack of sprouting angiogenesis during metastatic growth in lymph nodes using both intravital microscopy and ex vivo methods. Vascular endothelial growth factor was not produced by the metastatic lesion despite the presence of hypoxia in some lesions. Treatment with two different antiangiogenic therapies showed no effect on the growth or vascular density of lymph node metastases in our models. We confirmed these findings in clinical specimens, including the lack of reduction in blood vessel density in lymph node metastases in patients treated with bevacizumab. Conclusion: Tumors can impair the function of tumor draining lymphatic vessels, which may limit cancer cell dissemination as well the initiation of anti-tumor immunity. We provide pre-clinical and clinical evidence that sprouting angiogenesis does not occur during the growth of lymph node metastases, which reveals a mechanism of treatment resistance to antiangiogenic therapy in adjuvant settings. The targets of clinically approved angiogenesis inhibitors are not active during early cancer progression in the lymph node, suggesting that inhibitors of sprouting angiogenesis as a class will not be effective in treating lymph node metastases. Citation Format: Timothy P. Padera. Lymphatic metastasis and the lymph node microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA14.

TNF‐α Inhibits Rat Mesenteric Lymphatic Pumping via Activation of the NF‐κB and Upregulation of iNOS

Upregulated TNF‐α production and dysfunctional lymphatic pumping are observed in inflammatory bowel disease pathogenesis. It has been previously demonstrated that TNF‐α could decrease arterial contractility via activation of the inducible nitric oxide synthase (iNOS) and that iNOS is involved in impaired mesenteric lymphatic pumping in animal model of intestinal inflammation. We investigated the effects of TNF‐α on rat mesenteric lymphatic pumping and hypothesized that the cytokine may impair lymphatic contractile function during intestinal inflammation by upregulating the synthesis of iNOS and thus the production of nitric oxide.Using pressure myography, we examined the contractile function of isolated rat mesenteric lymphatic vessels following a 24‐hr treatment with TNF‐α. We observed that contraction frequency was significantly and concentration‐dependently decreased by TNF‐α, an effect restored following administration of inhibitors of NF‐κB (PDTC), iNOS (1400W), guanylate synthase (ODQ) and ATP‐sensitive K+ channels (glibenclamide), but not the cyclooxygenase inhibitor, indomethacin. The involvements of NF‐κB and iNOS were further confirmed by western blot analysis and quantitative real‐time PCR, which revealed upregulations of phospho‐IκB and iNOS mRNA in TNF‐α‐treated vessels that were minimized in the presence of PDTC.These findings suggest that TNF‐α decreases lymphatic contractility via activation of the NF‐κB‐iNOS pathway.

Dextran sulfate sodium-induced acute colitis impairs dermal lymphatic function in mice.

To investigate whether dermal lymphatic function and architecture are systemically altered in dextran sulfate sodium (DSS)-induced acute colitis. Balb/c mice were administered 4% DSS in lieu of drinking water ad libitum for 7 d and monitored to assess disease activity including body weight, diarrhea severity, and fecal bleeding. Control mice received standard drinking water with no DSS. Changes in mesenteric lymphatics were assessed following oral administration of a fluorescently-labelled fatty acid analogue, while dermal lymphatic function and architecture was longitudinally characterized using dynamic near-infrared fluorescence (NIRF) imaging following intradermal injection of indocyanine green (ICG) at the base of the tail or to the dorsal aspect of the left paw prior to, 4, and 7 d after DSS administration. We also measured dye clearance rate after injection of Alexa680-bovine serum albumin (BSA). NIRF imaging data was analyzed to reveal lymphatic contractile activity after selecting fixed regions of interest (ROIs) of the same size in fluorescent lymphatic vessels on fluorescence images. The averaged fluorescence intensity within the ROI of each fluorescence image was plotted as a function of imaging time and the lymphatic contraction frequency was computed by assessing the number of fluorescent pulses arriving at a ROI. Mice treated with DSS developed acute inflammation with clinical symptoms of loss of body weight, loose feces/watery diarrhea, and fecal blood, all of which were aggravated as disease progressed to 7 d. Histological examination of colons of DSS-treated mice confirmed acute inflammation, characterized by segmental to complete loss of colonic mucosa with an associated chronic inflammatory cell infiltrate that extended into the deeper layers of the wall of the colon, compared to control mice. In situ intravital imaging revealed that mice with acute colitis showed significantly fewer fluorescent mesenteric lymphatic vessels, indicating impaired uptake of a lipid tracer within mesenteric lymphatics. Our in vivo NIRF imaging data demonstrated dilated dermal lymphatic vessels, which were confirmed by immunohistochemical staining of lymphatic vessels, and significantly reduced lymphatic contractile function in the skin of mice with DSS-induced acute colitis. Quantification of the fluorescent intensity remaining in the depot as a function of time showed that there was significantly higher Alexa680-BSA fluorescence in mice with DSS-induced acute colitis compared to pre-treatment with DSS, indicative of impaired lymphatic drainage. The lymphatics are locally and systemically altered in acute colitis, and functional NIRF imaging is useful for noninvasively monitoring systemic lymphatic changes during inflammation.