Niklas Telinius

Human thoracic duct in vitro: diameter-tension properties, spontaneous and evoked contractile activity

The current study characterizes the mechanical properties of the human thoracic duct and demonstrates a role for adrenoceptors, thromboxane, and endothelin receptors in human lymph vessel function. With ethical permission and informed consent, portions of the thoracic duct (2-5 cm) were resected and retrieved at T(7)-T(9) during esophageal and cardia cancer surgery. Ring segments (2 mm long) were mounted in a myograph for isometric tension (N/m) measurement. The diameter-tension relationship was established using ducts from 10 individuals. Peak active tension of 6.24 +/- 0.75 N/m was observed with a corresponding passive tension of 3.11 +/- 0.67 N/m and average internal diameter of 2.21 mm. The equivalent active and passive transmural pressures by LaPlaces law were 47.3 +/- 4.7 and 20.6 +/- 3.2 mmHg, respectively. Subsequently, pharmacology was performed on rings from 15 ducts that were normalized by stretching them until an equivalent pressure of 21 mmHg was calculable from the wall tension. At low concentrations, norepinephrine, endothelin-1, and the thromboxane-A(2) analog U-46619 evoked phasic contractions (analogous to lymphatic pumping), whereas at higher contractions they induced tonic activity (maximum tension values of 4.46 +/- 0.63, 5.90 +/- 1.4, and 6.78 +/- 1.4 N/m, respectively). Spontaneous activity was observed in 44% of ducts while 51% of all the segments produced phasic contractions after agonist application. Acetylcholine and bradykinin relaxed norepinephrine preconstrictions by approximately 20% and approximately 40%, respectively. These results demonstrate that the human thoracic duct can develop wall tensions that permit contractility to be maintained across a wide range of transmural pressures and that isolated ducts contract in response to important vasoactive agents.

The human thoracic duct is functionally innervated by adrenergic nerves

Lymphatic vessels from animals have been shown to be innervated. While morphological studies have confirmed human lymphatic vessels are innervated, functional studies supporting this are lacking. The present study demonstrates a functional innervation of the human thoracic duct (TD) that is predominantly adrenergic. TDs harvested from 51 patients undergoing esophageal and cardia cancer surgery were either fixed for structural investigations or maintained in vitro for the functional assessment of innervation by isometric force measurements and electrical field stimulation (EFS). Electron microscopy and immunohistochemistry suggested scarce diffuse distribution of nerves in the entire vessel wall, but nerve-mediated contractions could be induced with EFS and were sensitive to the muscarinic receptor blocker atropine and the α-adrenoceptor blocker phentolamine. The combination of phentolamine and atropine resulted in a near-complete abolishment of EFS-induced contractions. The presence of sympathetic nerves was further confirmed by contractions induced by the sympathomimetic and catecholamine-releasing agent tyramine. Reactivity to the neurotransmitters norepinephrine, substance P, neuropeptide Y, acetylcholine, and methacholine was demonstrated by exogenous application to human TD ring segments. Norepinephrine provided the most consistent responses, whereas responses to the other agonists varied. We conclude that the human TD is functionally innervated with both cholinergic and adrenergic components, with the latter of the two dominating.

Identification of Interstitial Cajal-Like Cells in the Human Thoracic Duct

Interstitial Cajal-like cells (ICLCs) are speculated to be pacemakers in smooth muscle tissues. While the human thoracic duct (TD) is spontaneously active, the origin of this activity is unknown. We hypothesized that ICLCs could be present in the TD and using histological techniques, immunohistochemistry and immunofluorescence we have investigated the presence of ICLCs, protein markers for ICLCs and the cellular morphology of the human TD. Transmission electron microscopy was employed to investigate ultrastructure. Methylene blue staining, calcium-dependent fluorophores and confocal microscopy were used to identify ICLCs in live tissue. Methylene blue stained cells with morphology suggestive of ICLCs in the TD. Immunoreactivity localized the ICLC protein markers c-kit, CD34 and vimentin to many cells and processes associated with smooth muscle cells (SMCs): coexpression of c-kit with vimentin or CD34 was observed in some cells. Electron microscopy analysis confirmed ICLCs as a major cell type of the human TD. Lymphatic ICLCs possess caveolae, dense bands, a patchy basal lamina, intermediate filaments and specific junctions to SMCs. ICLCs were ultrastructurally differentiable from other interstitial cells observed: fibroblasts, mast cells, macrophages and pericytes. Lymphatic ICLCs were localized to the subendothelial region of the wall as well as in intimate association with smooth muscle bundles throughout the media. ICLCs were morphologically distinct with multiple processes and also spindle shapes. Confocal imaging with calcium-dependent fluorophores corroborated cell morphology and localization observed in fixed tissues. Lymphatic ICLCs thus constitute a significant cell type of the human TD and physically interact with lymphatic SMCs.

Lymph vessels: the forgotten second circulation in health and disease

The lymphatic circulation is still a somewhat forgotten part of the circulatory system. Despite this, novel insights in lymph angiogenesis in health and disease, application of immune markers for lymphatic growth and differentiation and also the introduction of new imaging techniques to visualize the lymphatic circulation have improved our understanding of lymphatic function in both health and disease, especially in the last decade. These achievements yield better understanding of the various manifestations of lymph oedemas and malformations, and also the patterns of lymphovascular spread of cancers. Immune markers that recognize lymphatic endothelium antigens, such as podoplanin, LYVE-1 and Prox-1, can be successfully applied in diagnostic pathology and have revealed (at least partial) lymphatic differentiation in many types of vascular lesions.

Voltage‐gated sodium channels contribute to action potentials and spontaneous contractility in isolated human lymphatic vessels

Initial studies have described the electrical properties of lymphatic smooth muscle cells from different animal species and the ion channels contributing to these properties. However, there is a translational gap to the human situation where studies on human tissue are lacking. Voltage‐gated sodium channels are essential for the generation of action potentials, and thus spontaneous contractions in human lymphatic vessels, but not noradrenaline‐induced contractions. A sodium channel opener elicited contractions as a result of calcium influx via voltage‐gated calcium channels and the sodium–calcium exchanger. Pharmacological characterization and the mRNA expression profile indicate that Nav1.3 is the most prevalent sodium channel. These results provide support for an important role of sodium channels in spontaneous human lymphatic vessel electrical activity and contractility.

Protection against high intravascular pressure in giraffe legs

The high blood pressure in giraffe leg arteries renders giraffes vulnerable to edema. We investigated in 11 giraffes whether large and small arteries in the legs and the tight fascia protect leg capillaries. Ultrasound imaging of foreleg arteries in anesthetized giraffes and ex vivo examination revealed abrupt thickening of the arterial wall and a reduction of its internal diameter just below the elbow. At and distal to this narrowing, the artery constricted spontaneously and in response to norepinephrine and intravascular pressure recordings revealed a dynamic, viscous pressure drop along the artery. Histology of the isolated median artery confirmed dense sympathetic innervation at the narrowing. Structure and contractility of small arteries from muscular beds in the leg and neck were compared. The arteries from the legs demonstrated an increased media thickness-to-lumen diameter ratio, increased media volume, and increased numbers of smooth muscle cells per segment length and furthermore, they contracted more strongly than arteries from the neck (500 ± 49 vs. 318 ± 43 mmHg; n = 6 legs and neck, respectively). Finally, the transient increase in interstitial fluid pressure following injection of saline was 5.5 ± 1.7 times larger (n = 8) in the leg than in the neck. We conclude that 1) tissue compliance in the legs is low; 2) large arteries of the legs function as resistance arteries; and 3) structural adaptation of small muscle arteries allows them to develop an extraordinary tension. All three findings can contribute to protection of the capillaries in giraffe legs from a high arterial pressure.

Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine

We studied the effects of antihypertensive calcium channel blockers on CaV1.2, the predominantly expressed L‐type calcium channel in the largest human lymphatic vessel, the thoracic duct. Human lymphatic collecting vessels, both large and small, are highly‐sensitive in vitro to calcium channel blockers; exposure to these drugs inhibits endogenous lymphatic contractile activity and action potentials and diminishes noradrenaline‐induced phasic contractions. In vivo administration of calcium channel blocker nifedipine to healthy volunteers did not reduce lymphatic contractile activity despite all subjects achieving nifedipine plasma concentrations comparable with those observed to affect contractile function in vitro. These results indicate that calcium channel blocker‐related oedema is unlikely to be exacerbated by an off‐target effect of the drugs diminishing lymphatic pumping and fluid removal.

Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride

Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl- is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl- gradient and Cl- channels were investigated by substitution of extracellular Cl- with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca2+-activated Cl- channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. About 100-300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl- movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl- being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca2+-activated Cl- channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.

Tone regulation in giraffe retinal arterioles.

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