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

Abstract

Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4 ± 4.9% of control in the presence of 20 nmol l⁻¹ nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the Ca_V1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo.

Introduction

Calcium channel blockers (CCB) of the dihydropyridine (DHP) class are one of the initial drug choices for hypertension treatment because of their powerful and consistent antihypertensive effect. DHPs inhibit calcium influx via L-type calcium channels into arterial smooth muscle cells (ASMC), thereby decreasing arterial tone, which reduces peripheral resistance and lowers blood pressure. DHPs (e.g. nifedipine, amlodipine) target the arteries selectively over the heart because of an 8- to 10-fold higher DHP sensitivity of the L-type calcium channel variants expressed in ASMC compared to cardiomyocytes (Welling et al. 1997; Zühlke et al. 1998).

One of the most common side-effects of DHP treatment is peripheral oedema. The oedema is dose dependent and occurs in approximately 9–30% of patients according to large clinical trials with DHPs (Hansson et al. 1999; Brown et al. 2000; Dahlöf et al. 2005; Philipp et al. 2007; Makani et al. 2011). Oedema is, however, rarely seen in patients treated with CCB from the phenylalkamine class (e.g. verapamil; Anderson et al. 1999). Although not life-threatening, oedema negatively influences the quality of life of affected individuals (Moffatt et al. 2003). The oedema is typically resistant to diuretics and, due to limited options for managing the oedema, 25% of the patients withdraw from the DHP treatment (Makani et al. 2011). The oedema is thought to occur due to a preferential arteriolar rather than venous dilatation leading to increased hydrostatic pressure in the capillaries and consequent increased extravasation of fluid, as dictated by the Starling forces (Messerli, 2001; Weir et al. 2001; Sica, 2003; Handler, 2004). The current hypothesis of DHP-induced oedema has focused only on one aspect of tissue fluid dynamics – the input (fluid filtration) – while the output (interstitial fluid removal by the lymphatic vasculature) has not been considered in this scenario. The lymphatic removal of interstitial fluid depends upon intrinsic contractions in the collecting lymphatic vessels, which pump the fluid back into the blood circulation in the great veins of the neck.

Voltage-gated calcium channels (VGCCs) have been determined to be critical for lymphatic contractile function in guinea-pigs (von der Weid et al. 2008), cows (Atchison & Johnston, 1997) and sheep (Beckett et al. 2007). Since an increase in fluid filtration requires well-functioning lymphatic vessels to maintain a normal interstitial fluid level and preliminary reports describe the importance of calcium channels in lymphatic contractile function, it is possible that the lymphatic fluid removal system is compromised by DHP treatment. It is therefore relevant to examine in detail the effect of DHP on human lymphatic vessels in vitro and in vivo. Near-infrared fluorescence imaging is a technique used to assess lymphatic pumping in humans (Unno et al. 2007; Sevick-Muraca et al. 2008) but has not yet been used in any drug trial. In this study we investigate whether, and to what degree, human lymphatic vessel contractile function in vitro is dependent on L-type calcium channel activity and characterize the sensitivity to the DHP CCB nifedipine and the non-DHP CCB verapamil. Finally, we investigate in a clinical trial what effect nifedipine administration to humans has on lymphatic function in vivo.

Methods

Ethical approval

Clinical trial Lymphatic dysfunction as a cause of calcium channel blocker oedema (EudraCT number: 2012-002622-75) was approved by the Danish Health and Medicine Agency, The Central Denmark Region Committees on Health Research Ethics and the Danish Data Protection Agency. The trial was monitored by the Good Clinical Practice unit at Aarhus University Hospital. All subjects participated voluntarily and with informed consent. Tissue (thoracic ducts and mesentery) was harvested from patients with informed consent and after approval from the Central Denmark Region Committees on Health Research Ethics and the Danish Data Protection Agency. Both the clinical trial and tissue harvest was conducted in accordance with the ethical principles for medical research involving human subjects as specified in the Helsinki declaration.

In vitro experiments

Tissue preparation

Thoracic ducts (TD) and mesenteric lymphatic (ML) vessels were harvested and used in this study. Thoracic ducts were harvested from patients undergoing oesophageal and cardiac cancer surgery (n = 55, age span 41–85 years) at the Department of Cardiothoracic Surgery, Aarhus University Hospital, Denmark. Tissue harvest was performed as described previously Telinius et al. 2010, 2014; Briggs Boedtkjer et al. 2013). A small piece of mesentery from jejunum was harvested from patients (n = 10, age span 33–45 years) undergoing bariatric surgery at Department of Surgery, Viborg Hospital, Denmark. Mesenteric lymphatic vessels were identified with a stereomicroscope and dissected free from surrounding tissue.

Isometric force measurements

Ring segments (2 mm long) of TD and ML were prepared and mounted on 40 μm wires in multichannel myographs (DMT) for isometric force recordings. The preparation, mounting, and experiments were performed in physiological saline solution (PSS; for composition see Solutions below). The vessels were maintained at 37°C in PSS equilibrated with a mixture of 20% O₂ and 5% CO₂ balanced with N₂ throughout the experiments (pH 7.4). Isometric force development was recorded at 40 Hz with a Powerlab 4/25 system (ADInstruments) using LabChart software. Data files were saved for offline analysis. Force data were converted to tension (N m⁻¹) by dividing the force (mN) by two times the segment length (mm) (Mulvany & Halpern, 1977). Diameter was calculated using the following equation:

[(Total wire circumference + (2 × wire diameter)) + (2 × the distance between the inner edges of the wires)]/π.

Contraction frequency and amplitude of spontaneous activity was extracted from all experiments except when stimulation with noradrenaline (NA) or 123.7 mmol l⁻¹ KCl was used, in which case average tension during the stimulation period was extracted.

After mounting, the TD ring segments were stepwise stretched to a diameter at which the wall tension was equivalent to 20.6 mmHg (Telinius et al. 2010), allowing maximal force production. Following normalization the vessels were set to equilibrate for 30–60 min.

Thoracic duct ring segments were used for the following pharmacological protocols:

1. Exposure to 5 nmol l⁻¹, 20 nmol l⁻¹ and 100 nmol l⁻¹ of nifedipine (n = 15).

2. A cumulative concentration–response curve protocol for nifedipine (0.1 nmol l⁻¹–1 μmol l⁻¹; n = 7) or verapamil (0.1 nmol l⁻¹–3 μmol l⁻¹; n = 8), with half-logarithmic increases every 20 min.

3. A cumulative concentration–response curve protocol for noradrenaline (1 nmol l⁻¹–10 μmol l⁻¹; n = 9), with half-logarithmic increases very fifth minute, in the presence of 20 nmol l⁻¹ nifedipine.

4. Incubation with 123.7 mmol l⁻¹ KCl in the presence of 20 nmol l⁻¹ or 1 μmol l⁻¹ nifedipine (n = 6).

Spontaneously active mesenteric ring segments were exposed to a cumulative concentration–response curve protocol for nifedipine (0.1 nmol l⁻¹–1 μmol l⁻¹; n = 8) and verapamil (0.1 nmol l⁻¹–3 μmol l⁻¹; n = 3), with half-logarithmic increases every 20 min.

Membrane potential was measured simultaneously with force in three vessels exposed to the same nifedipine concentration–response curve.

Membrane potential measurements

Ring segments of ML were mounted in a single chamber myograph (DMT), normalized to 22 mmHg and allowed to equilibrate for 30 min, by which time spontaneous contractions had developed. Membrane potentials of lymphatic smooth muscle cells (LSMCs) were measured using sharp glass microelectrodes (WPI; AS100F) pulled on a horizontal puller (Sutter P-97) and then connected to an amplifier (WPI; Intra 767). The microelectrodes were filled with 3 mol l⁻¹ KCl and were flexible enough to maintain recording from a single LSMC even during depolarization and activation. A successful penetration and recording were defined by: (1) rapid, negative change in potential upon penetration (2) rapid positive deflection upon withdrawal of the electrode from the cell (3) unchanged resistance (40–120 MΩ) throughout the recording. The reference electrode was fixed and placed in the myograph chamber. Signal acquisition was performed at 1 kHz.

Expression analysis for L-type calcium channels using RT-PCR

After dissection, segments of human TD (eight patients) were placed in RNAlater (Sigma-Aldrich, Brøndby, Denmark) and thereafter stored at −20°C until RNA isolation. Samples were initially disrupted using a Tissue Lyser (Qiagen, Qiagen Nordic, Copenhagen, Denmark) for 3 min at 30 Hz before using the RNeasy Mini Kit and QIAcube system (Qiagen) to isolate RNA according to the manufacturer's protocol. First strand synthesis was performed using random decamers and SuperScript III reverse transcriptase (Invitrogen, Life Technologies Europe BV, Nærum, Denmark) with Superase treatment to deactivate RNase activity. The cDNA was amplified in an RT-PCR reaction using a Primus96 plus thermal cycler (PeqLab) according to the following protocol: initial activation of Ex Taq HS DNA polymerase (TaKaRa, Th. Geyer Denmark, Roskilde, Denmark) and DNA denaturation at 95°C for 4 min followed by 35 amplification cycles of 10 s at 95°C, 20s at 55°C and 30 s at 72°C. The following primers were used: CACNA1S (Ca_V1.1: forward 5′–3′cagtcggagcagatgaacca and reverse 5′–3′ gcaaagttggtgccacatgtgt), CACNA1C (Ca_V1.2: forward 5′–3′ cggcatcaccaactttgacaac and reverse 5′–3′ ccttggccttctccctctc) CACNA1D (Ca_V1.3: forward 5′–3′ caagatgttcaatgatgccatgga and reverse 5′–3′ ctggttgttatctctcatggcaac) and for RNA integrity purposes TFRC (forward 5′–3′ cgcagaactttcattctttggac and reverse 5′–3′ ctgggcaagtttcaataggaga). Repeated attempts to amplify CACNA1F (Ca_V1.4) from the control expression tissues (human retinal pigment epithelium total RNA; ScienCell Research Laboratories, and human skeletal muscle and adrenal gland total RNA; Stratagene, AH Diagnostics, Aarhus V, Denmark) – with primers based closely upon published human primers (McRory et al. 2004); forward 5′–3′ ccaatgcctgctactgggc and reverse 5′–3′ gagatgcccaagggctgc) – failed, despite positive TFRC amplification, so this isoform was not pursued with patient RNA. PCR products were loaded on a 2% TBE agarose gel then separated by electrophoresis in TBE buffer and visualized using Midori Green DNA stain (Nippon Genetics, Kem-En-Tec Nordic A/S, Taastrup, Denmark) or ethidium bromide with UV illumination (Gel Doc 2000; BioRad). Primer synthesis and band sequencing were performed by Eurofins Genomics (Germany). Control total RNA sources used as controls were human skeletal muscle (Ca_V1.1) and human carotid artery (Ca_V1.2) (Stratagene, Agilent Technologies) and human brain (Stratagene) or dorsal root ganglion (Ca_V1.3) (Clontech).

Whole-mount immunofluorescence

The whole-mount immunofluorescence method has been described previously (Briggs Boedtkjer et al. 2013). Briefly, 4% paraformaldehyde-fixed human thoracic duct tissue was washed and stored in phosphate-buffered saline (PBS) at 4°C until use. After an initial wash in new PBS, duct tissue was blocked and permeabilized in a solution of PBS containing 1.5% bovine serum albumin and 0.3% Triton X-100, which was used throughout the rest of the protocol. The tissue was then incubated overnight at 4°C on a shaking table in the primary antibody solution (1:100 rabbit polyclonal anti-Ca_V1.2; Alomone Labs, ACC-003). Controls were performed in parallel with either the primary antibody omitted or with primary antibody preincubated with the antigenic peptide. The following day the tissue was washed three times (15 min duration each) before incubating with the secondary antibody (1:1000 goat anti-rabbit F(ab′)₂ Alexa Fluor 546; Invitrogen, A11071) for 2 h in the dark at room temperature. Subsequently the tissue was washed three times in the dark and finally incubated for 10 min in 1 μmol l⁻¹ SYTO 16 (Invitrogen, S7578) to co-stain nuclei. Duct tissue was mounted on slides and imaged using a Zeiss LSM5 Pascal confocal microscope using 40× water-immersion objective and the appropriate laser and filter sets.

Solutions and chemicals

Control RNA was purchased from AH Diagnostics (Aarhus, Denmark). All salts and drugs were purchased from Sigma-Aldrich. Physiological saline solution of the following composition was used (in mmol l⁻¹): 119 NaCl, 4.7 KCl, 1.17 MgSO₄, 25 NaHCO₃, 1.18 KH₂PO₄, 0.026 EDTA, 5.5 glucose and 1.6 CaCl₂. All drugs were dissolved in distilled water, except for nifedipine (ethanol), and were stored in aliquots at −20°C until required. All drugs were diluted in PSS. At the maximum nifedipine concentration (1 μmol l⁻¹), the concentration of ethanol in the bath was 0.01% (v/v). The bath concentration of ethanol never exceeded this level. Furthermore, lower concentrations of nifedipine had commensurately lower concentrations of ethanol. PBS was composed of 55 mmol l⁻¹ Na₂HPO₄, 13 mmol l⁻¹ NaH₂PO₄, and 57 mmol l⁻¹ NaCl adjusted to pH 7.50 with NaOH.

Near-infrared fluorescence imaging of lymphatic vessels in humans

Room temperature was measured and noted continuously throughout the investigation and maintained within a 2°C range. The start time for each image sequence was noted and on the second visit each image series was acquired on approximately the same time points as on the first visit. All test days started between 0.800 and 0.900 h to eliminate any diurnal changes in the interstitial fluid balance. The subjects had refrained from alcoholic beverages and physical exercise for the previous 24 h and xanthine-containing beverages for previous 12 h.

Lymphatic vessels on the foot and calf were investigated using near-infrared fluorescence (NIRF) imaging. Images were obtained by a Hamamatsu C9100-13 EM-CCD camera with a Navitar 25 mm f0.95 lens with two 835 nm ± 15 nm (> OD5) band-pass filters mounted in front and behind. As a light source we used a PowerTechnology 450 mW 785 nm laser with a 780 nm ± 28 nm band-pass filter and a lens to spread the light. Indocyanine green (ICG; Nomeco, Denmark) was dissolved in sterile water and diluted with isotonic saline to a final concentration of 0.3 g l⁻¹. Indocyanine green (0.1 ml; 30 μg) was injected intradermally with 31G needles (Wiotech, Denmark) in the interdigital space between the first and second toe and the fourth and fifth toe as well as behind the medial malleolus. The subjects were in a supine position throughout the investigations. The first image sequence was started approximately 20 min after injection. In total seven separate sequences were acquired in the following order: foot (Supporting information Movie S1, Photograph S1), lower leg (Supporting information Movie S2, Photograph S2; Fig.6), refill time (Supporting information Movie S3, Photograph S3; Fig.7), pumping pressures (Supporting information Movie S4, Fig.8), foot, lower leg and refill time (with the last three acquired after a one-hour 40°C foot bath). All image sequences started with the subject lying still for 3 min. Foot and leg sequences were 8 min long where the foot or leg was completely still (referred to as inactivity) for the first 4 min and the last 4 min the subject made an extension–flexion movement of the toes every 30 s (referred to as activity). Refill time sequences were performed by placing a tourniquet a few centimetres above the malleolus to block lymph flow for 1 min while the vessels were emptied of lymph by manual massage. Subsequently, the tourniquet was removed and the vessels were allowed to refill. This was repeated three times. Pumping pressure were measured by emptying the vessels for lymph as described above; however, prior to removing the tourniquet a cuff was placed proximally to the tourniquet and inflated to 70 mmHg (Hokanson E20 Rapid cuff inflator, Hokanson AG101 air source, SC10 cuff). The cuff pressure was lowered 5 mmHg every fifth minute until lymph was observed to cross the cuff and flow resumed.

Figure 6. Near-infrared fluorescence imaging with indocyanine green enables lymphatic contractile function to be measured in vivo.

The image series depicts a network of lymphatic vessels on the lower leg. An example of a packet of lymph moving cranially is marked with a white arrow. Top right is a photograph of the same area. Bottom right shows an intensity plot over a longer time course for ROI A, B and C. Pulsatile changes in fluorescence intensity (FI) can be seen, reflecting lymphatic contractions. See Movie S2 in Supporting information.

Figure 7. Near-infrared fluorescence imaging with indocyanine green demonstrating how refill time is measured.

In the image time-stamped 0 s lymphatic vessels filled with fluorescent lymph can be seen. At 96 s a tourniquet (pointed out by white arrow) has been placed around the leg and the lymph has been manually removed using drainage massage. At 190 s the tourniquet has been released and one lymphatic vessel has so far been refilled halfway (asterisk). At 237 s the second vessel has also been refilled halfway (2 asterisks). Finally at 292 s all vessels are completely refilled. The intensity plot for ROI A and B is displayed at the bottom, FI = fluorescence intensity. When the lymph reaches the ROI, a clear increase in intensity occurs and subsequently pulses of lymph can be seen to propagate through the ROIs. See Movie S3 in Supporting information.

Figure 8. Near-infrared fluorescence imaging with indocyanine green enables lymphatic pumping pressures to be measured.

The image sequence depicts how lymphatic pumping pressure is determined. First, the existing fluorescent lymph has been removed (as described in Fig.8) and an inflatable cuff has been placed around the leg to obstruct lymph flow. The bright area marked by the rectangle is the inflated cuff. No lymph is seen cranial to the cuff. When the pressure is reduced from 65 mmHg to 60 mmHg at 339 s, the first fluorescent lymph packet (marked by an asterisk) soon crosses the cuff (image 418 s). Eventually more lymph transverses the cuff region (image 447 s) and new vessels also become apparent (image 480 s, double asterisk). The maximal pressure the lymphatic pump can exceed is thus 60 mmHg in this subject. See Movie S4 in Supporting information.

Quantification of lymphatic function

Image sequences were analysed in ImageJ (NIH, Bethesda, USA) and LabChart (ADInstrument). Regions of interest (ROIs) were placed on the vessels and intensity was measured throughout the sequence. A contraction was defined as a 10% increase in intensity and a visual confirmation of the image sequence that a lymph packet moved through the ROI. On the leg, the dominant vessels draining the injection sites on the foot and the medial malleolus was analysed: the dominant vessel was defined as the vessel with the highest contraction frequency. Packet velocity was calculated by measuring the time difference between the lymph packet occurring in two different ROIs placed on the vessel and dividing the distance between the ROIs (average distance: 7.3 ± 1.5 cm) by the time difference. Contraction frequencies, contraction velocities and refill times presented are the average of the two dominant vessels. On the foot, similarly to the leg, the dominant vessels draining the medial and lateral injections were determined and used in the same way as on the leg. The network of lymphatic vessels was almost identical on both test days, enabling us to place the ROIs on the same vessels on both test days.

Clinical trial: Lymphatic dysfunction as a cause of calcium channel blocker oedema

The study was designed as a randomized, double-blinded, placebo-controlled crossover trial (EudraCT number: 2012-002622-75). Inclusion criteria were male gender and age between 18 and 35 years. Exclusion criteria were arterial hypotension, orthostatic hypotension, angina pectoris, previous acute myocardial infarction, previous gastrointestinal bleeding, current treatment with any type of calcium channel blocker and allergy against ingredients in the tablets. All participants were assessed for eligibility and included by N.T. The study population was set to six subjects. Each subject went through two 12-day periods of administration of either nifedipine or placebo, with a washout period of 4 weeks between the end of the first period and the beginning of the second period. Subjects were randomly chosen to either start with nifedipine or placebo, with half of the group starting with placebo and the other half starting with nifedipine. During the 12-day periods the subjects were instructed to take one capsule daily for the first 3 days, two capsules daily the following 3 days and three capsules daily for the last 6 days. All capsules were taken at the same time of day. Each capsule contained either 30 mg Nifedipine Alternova or placebo. The final dose was 90 mg nifedipine daily, which was administered for 6 days. The treatment regime was not designed to precisely mimic clinical practice but to permit a high-dose regimen for nifedipine to be achieved within a short time frame and with sufficient dosing to reach a pharmacological steady state. Nifedipine and placebo tablets were encapsulated to blind the subject and investigator. Aarhus University Hospital Pharmacy performed the randomization and blinding. The blinding was removed after all data were analysed. Before the 12-day administration period was initiated, measurements of blood pressure, pulse, body weight and foot volume were obtained. The same measurements were repeated after the completion of the 12-day period and NIRF imaging of the lymphatic vessels on the foot and lower leg was performed. A blood sample was also taken to determine the plasma concentration of nifedipine. Primary end-points for the study were obtained using NIRF imaging and were: contraction frequency, contraction wave propagation velocity, maximal occlusion pressure preventing lymph flow and refill time. Secondary end-points were the same parameters after one immersion of the lower leg in a 40°C water bath. Secondary end-points were chosen to test lymphatic function after a period of vasodilatation, previously shown to increase lymph flow (Olszewski et al. 1977). The trial is reported according to the CONSORT 2010 guidelines for reporting a randomized trial. Secondary end-points could only be obtained in four subjects because of a loss of signal after the footbath (something that did not occur in pilot experiments). As a consequence these results were discarded and not presented.

Foot volume estimation

Foot volume was estimated volumetrically by water displacement. A water bath was filled to 25 cm with 30°C water after which the subject immersed and placed the foot on the bottom of the bath. The position of the foot was kept constant by a horizontal position bar placed at the opening of the bath. The water displaced by the foot was collected and weighed (OBH Nordica, Denmark) and corresponds to the volume of the foot. The measurement was repeated three times and an average was calculated. During one of subject 6's visits the bath was damaged and a leakage was discovered afterwards which forced us to exclude that individual from the foot volume measurements.

Blood pressure measurement

Blood pressure was measured in seated subjects on the left arm with a manual sphygmomanometer and the average of three measurements is reported.

Measurement of nifedipine in plasma

All blood samples were stored at −20°C until analysed. Nifedipine was extracted using a mixture of methanol and acetonitrile following protein precipitation. The extract was ultra-filtrated and analysed by ultra-high performance liquid chromatography mass spectrometry (UPLC-MS/MS). Deuterium-labelled nifedipine was used as internal standard and an 8-point calibration was performed using blood spiked with defined amounts of nifedipine reference material (Sigma-Aldrich catalogue number N7634). The calibration range was 3.9–500 μg l⁻¹ and the lower limit of quantitation was 2.6 μg l⁻¹. All sample preparation was performed with a minimum of light exposure because of nifedipine's photosensitivity. Method validation parameter calculations were based on recommendation provided by the Scientific Working Group for Forensic Toxicology (http://www.swgtox.org): Standard Practices for Method Validation in Forensic Toxicology. The current method is based on methodologies developed by Sørensen and Hasselstrøm (Sørensen & Hasselstrøm, 2013).

Statistics

Microsoft Excel was used for data storage and all statistical analyses were conducted in GraphPad Prism version 5. All in vivo data were analysed using a Student's paired t test. Single concentration effects from the myograph experiments were analysed using Student's paired t test. Concentration–response curves to NA were analysed by least-squares fitting to a sigmoidal curve and compared by extra sum-of-squares F test. Correlation between CCB treatment and spontaneous activity in vitro was analysed with a χ² test. All data are presented as means ± SEM. Significance level was set at P = 0.05.

Results

Ca_v1.2 is the predominant calcium channel expressed in human lymphatic vessels

Human thoracic duct RNA from eight individuals was investigated for the presence of the pore-forming α-1 subunit genes of L-type calcium channels; CACNA1S (Ca_V1.1), CACNA1C (Ca_V1.2) and CACNA1D (Ca_V1.3). A transcript for CACNA1C was amplified from all individuals (n = 8) examined by RT-PCR (Fig.1A, right). Interestingly, some individuals expressed CACNA1C alone (n = 2) or in combination with either CACNA1S (n = 2) or CACNA1D (n = 2), and in some instances all three isoforms were detected (n = 2). Sequence analysis of the RT-PCR bands confirmed the identities of the amplified products. The band for CACNA1S was consistently smaller than predicted for the reference sequence (NM_000069.2) and when sequenced was found to lack exon 29: skipping of this exon shortens the extracellular loop between the transmembrane domains IVS3 and IVS4 and has been reported to confer higher conductance and voltage sensitivity to the Ca_V1.1 Ca²⁺ channel in skeletal muscle (Tuluc et al. 2009). The Ca_V1.2 sequence translated to the amino acid sequence VNDAVGRDWPWIYFVTLIIIGSFFVLNLVLGVLSG, which corresponds to the cardiac isoform exon 8a. As CACNA1C was the consistently expressed isoform, we performed immunoreactivity experiments to localize the protein expression in whole-mount sections of human thoracic duct. Using confocal microscopy, the anti-Ca_V1.2 signal was localized to the LSMCs. Co-staining of nucleic acids with SYTO 16 revealed positive immunostaining for Ca_V1.2 in cells with typical cigar-shaped smooth muscle cell nuclei (Fig.1B). Z-stack images constructed through the duct wall confirmed the positive staining to be constrained to the media, with no Ca_V1.2 immunostaining observed in the endothelial cell layer. The parallel control experiments were negative (with tissue either incubated without the primary antibody or with the antibody preincubated with antigenic peptide; data not shown).

Figure 1. Ca_v1.2 is the predominantly expressed VGCC in human lymphatic vessels.

A, CACNA1C (Ca_V1.2) is consistently expressed at the mRNA level with variable or no expression of CACNA1S (Ca_V1.1) and CACNA1D (Ca_V1.3), while all patient and control samples were positive for the housekeeping control gene transferrin receptor (TFRC); 1–3 represent patients 1–3; SM, skeletal muscle; CA, carotid artery; Br, brain; 200 and 500 refer to base pair size for the DNA marker in lane 1. B, immunoreactivity for the CACNA1C protein is localized to smooth muscle cells in the media of the human thoracic duct (1:100 rabbit polyclonal anti-Ca_V1.2 and secondary goat anti-rabbit Alexa Fluor 546, nucleic acid staining with SYTO 16). Asterisks highlight cells with typical smooth muscle cell nuclei and cell morphology. The images are representative for four patients investigated. Scale bars represent 50 μm.

Spontaneous contractions in human lymphatic vessels are inhibited by therapeutic concentrations of nifedipine

We challenged TD ring segments mounted in a wire myograph with 5 nmol l⁻¹ nifedipine, which is the approximate plasma concentration achieved during chronic nifedipine treatment (Lederballe Pedersen et al. 1980; Stern et al. 1984; van Bortel et al. 1989; Cainazzo et al. 2005). Additionally we tested two higher concentrations (20 nmol l⁻¹ and 100 nmol l⁻¹). Spontaneous contractions in ring segments from 8 out of 15 patients ceased in the presence of 5 nmol l⁻¹ nifedipine. The number of active vessels was reduced to two with 20 nmol l⁻¹ nifedipine present and none were spontaneously active when 100 nmol l⁻¹ was present. To further characterize the sensitivity to nifedipine, as well as the effect of verapamil, we exposed TD and mesenteric lymphatic (ML) ring segments mounted in a wire myograph to increasing concentrations of nifedipine (0.1 nmol l⁻¹–1 μmol l⁻¹, in half-logarithmic steps) or verapamil (0.1 nmol l⁻¹–3 μmol l⁻¹, in half-logarithmic steps). Spontaneous lymphatic contractions were abolished by both nifedipine and verapamil: nifedipine exerted an effect within a few minutes whereas verapamil required up to 15 min for full effect consistent with its use-dependent mode of inhibition (Pelzer et al. 1982). While both drugs abolished spontaneous contractions, nifedipine did so in the nanomolar range as opposed to the micromolar concentrations of verapamil required for the same effect (Fig.2). In this second series of nifedipine experiments the concentration required to completely abolish phasic contractions (pIC₁₀₀) was 8.4 ± 0.2 and 8.5 ± 0.4 in TD and ML ring segments, respectively. The corresponding values for verapamil in TD and ML ring segments were 6.1 ± 0.1 and 6.2 ± 0.3, respectively, which are higher concentrations than achieved clinically during verapamil therapy. Simultaneous recordings of force and membrane potential from LSMCs showed that each spontaneous contraction was preceded by an action potential under normal conditions (Fig.3). Nanomolar concentrations of nifedipine abolished spontaneous action potentials and consequently the phasic contractions. Nifedipine (mean concentration 12 ± 1.6 nmol l⁻¹) furthermore significantly depolarized the vessels from a resting membrane potential (RMP) of −44 ± 3 mV to −37 ± 1 mV (P = 0.0426, paired Student's t test; Fig.3B).

Figure 2. Calcium channel blockers nifedipine and verapamil abolish phasic contractions of mesenteric and thoracic duct ring segments mounted in a wire myograph with different potency.

A, original data trace of a thoracic duct ring segment showing phasic contractions abolished by nanomolar concentrations of nifedipine. B, cumulative data for contraction frequency and amplitude of thoracic duct ring segments exposed to increasing concentrations of nifedipine and verapamil. C, corresponding cumulative results for mesenteric lymphatic vessels. D, plot showing the concentrations of verapamil and nifedipine in thoracic duct (TD) and mesenteric lymphatic (ML) ring segments that inhibit spontaneous phasic activity (IC₁₀₀). Data represent means ± SEM.

Figure 3. The calcium channel blocker nifedipine abolishes action potentials and depolarizes lymphatic smooth muscle cells in the thoracic duct.

A, simultaneous force and membrane potential measurement from a thoracic duct ring segment mounted in a wire myograph showing an abolishment of action potentials by 10 nmol l⁻¹ nifedipine. B, nifedipine consistently depolarized the membrane potential at the lowest concentration necessary to abolish phasic contractions and action potentials (n = 4). *P = 0.0426, paired Student's t test.

Calcium channel blocker nifedipine inhibits agonist-induced contractions

Having established that clinically relevant concentrations of nifedipine abolished spontaneous phasic contractions in human lymphatic vessels, we attempted to elucidate the role of the L-type VGCC for agonist-induced contractile responses. We have previously demonstrated the presence of a functional adrenergic innervation of the human thoracic duct and noradrenaline (NA) is a reliable and potent vasoconstrictor of this tissue (Telinius et al. 2010, 2013). Therefore we generated NA cumulative concentration–response curves (10 nmol l⁻¹–10 μmol l⁻¹) in the absence and presence of nifedipine. We found that nifedipine (20 nmol l⁻¹) significantly reduced the contractile response to the majority of NA concentrations (100 nmol l⁻¹–10 μmol l⁻¹; Fig.4): the maximal response to NA was reduced from 91.0 ± 5.4% (normalized to the initial 10 μmol l⁻¹ NA exposure) to 29.4 ± 4.9% (P < 0.0001, extra sum-of-squares F test). Furthermore, in the presence of nifedipine, NA only increased baseline tension and failed to elicit the phasic contractions observed in the absence of nifedipine (Fig.4). The NA-sensitivity of the tissue was unaffected by nifedipine: the pEC₅₀ in the presence of nifedipine (6.6 ± 0.2) was unchanged compared to control (6.7 ± 0.09; P = 0.7390, extra sum-of-squares F test). The effect of nifedipine was also evaluated on 125 mmol l⁻¹ K⁺-induced contractions: exposure to high concentrations of potassium causes a marked depolarization of cells that opens VGCC. Compared to the control K⁺-evoked contraction, preincubation with 20 nmol l⁻¹ nifedipine reduced the response to 61 ± 6% (P = 0.0176, paired Student's t test) and 1 μmol l⁻¹ reduced it further to 22 ± 4% (P = 0.0004, paired Student's t test; Fig.4).

Figure 4. Nifedipine abrogates noradrenaline-induced phasic contractions and tone in the thoracic duct.

A, a typical example of a noradrenaline cumulative concentration–response curve in the absence (left) and presence (right) of 20 nmol l⁻¹ nifedipine. B, cumulative concentration–response data showing a significant reduction in noradrenaline-stimulated tone in the presence of 20 nmol l⁻¹ nifedipine (bottom). **P < 0.0001, extra sum-of-squares F test, n = 9. C, contractions induced by elevating extracellular potassium (to 125 mmol l⁻¹ K⁺), to stimulate VGCC activity, are reduced by the presence of 20 nmol l⁻¹ and 1 μmol l⁻¹ nifedipine. *P < 0.05 compared to control, paired Student's t test, n = 6. Data represent means ± SEM.

Calcium channel blocker therapy does not affect spontaneous contractions in vitro

Not all TD segments harvested from patients exhibit spontaneous contractions in vitro (Sjoberg & Steen, 1991; Telinius et al. 2010). We analysed if there was a correlation between the observations of spontaneous activity in vitro and if the patient's normal medication included a DHP. In total 126 patients were included in the analysis and 27 of these were in a CCB treatment regimen (amlodipine; average dose 7 ± 0.5 mg). The data were analysed with a χ² test and showed no significant correlation between CCB treatment and spontaneous activity in vitro (P = 0.1899). Average contraction frequency and amplitude were similar: treated 1.2 ± 0.2 min⁻¹ and 2.5 ± 0.3 N m⁻¹, respectively, compared to untreated 1.2 ± 0.1 min⁻¹ and 3.0 ± 0.2 N m⁻¹, respectively.

Clinical trial: Lymphatic dysfunction as a cause of calcium channel blocker oedema

Six individuals were assessed for eligibility and included in the study. No participants were excluded or lost to follow-up. Participants were recruited in June–July 2013 and the trial was completed in November 2013. Baseline characteristics can be found in Table1. Two participants reported side-effects (flushing); one on nifedipine and one on placebo. Foot volume did not increase with nifedipine treatment. Haemodynamically, the mean arterial pressure was lowered in 5 out of 6 subjects and heart rate increased in 4 out of 6 patients; however, the average difference did not reach statistical significance (Table2). Variation in the haemodynamic measurements was noted when we compared the baseline measurements before nifedipine/placebo treatment, suggesting that a ‘white-coat’ effect contributed in some subjects. This limited the evaluation of the haemodynamic effects of nifedipine and the study was neither designed nor powered to take this into account. The mean plasma concentration of nifedipine was 75 ng ml⁻¹ (range: 21–148 ng ml⁻¹), equivalent to 11 nmol l⁻¹, and at the high end compared to previous studies (2–90 ng ml⁻¹; Lederballe Pedersen et al. 1980; Kleinbloesem et al. 1987b; Akopov et al. 1994; Cainazzo et al. 2005).

Table 1.

Study population demographics

Baseline characteristics
BMI (kg m−2)	22 ± 0.5 (20–24)
Age (years)	26 ± 0.4 (24–27)
Systolic blood pressure (mmHg)	132 ± 4 (116–139)
Diastolic blood pressure (mmHg)	80 ± 5 (58–91)
Pulse (beats min–1)	62 (41–73)

Data are means ± SEM (range).

Table 2.

Non-lymphatic parameters

Measurement	Placebo	Nifedipine	P value
ΔMean arterial pressure (mmHg)	0.3 ± 3	−5.9 ± 4	0.1802
ΔPulse (beats min–1)	1.6 ± 3	0.7 ± 6	0.8708
ΔFoot volume (ml)	−28 ± 31	−5 ± 39	0.6767
ΔBody weight (kg)	0.8 ± 0.5	−1 ± 0.5	0.1173

Data for placebo and nifedipine are means ± SEM (range).

NIRF imaging of the lymphatic vasculature on the foot and leg revealed a network of lymphatic vessels, with pulsatile changes in fluorescence intensity propagating proximally, reflecting periodic lymphatic contractions (Supporting information Movies S1 and S2). The branching and number of vessels varied between subjects. The network appearance on the leg was essentially unchanged on both test days, which enabled us to analyse regions of interest in the same location in both data sets. We analysed the dominant vessel – defined as the vessel with highest contraction frequency – that drained the injections on the foot and behind the medial malleolus. The dominant vessel was the same on both test days.

Primary end-points

Leg

We found that contraction frequency in the lower leg lymphatics was higher when the leg was static (defined as inactivity) during nifedipine administration compared to placebo: 1.05 ± 0.16 min⁻¹ versus 0.77 ± 0.15 min⁻¹ (P = 0.0350, paired Student's t test, n = 6; Fig.5A). Contraction frequency in the same vessels during activity (gentle extension of the foot every 30 s) was not statistically different (1.6 ± 0.3 min⁻¹ versus 1.1 ± 0.2 min⁻¹) but showed a tendency towards confirming the finding during inactivity (P = 0.0688, paired Student's t test, n = 6). The vessels’ refill time was significantly shorter in the nifedipine period compared to placebo: 76 ± 9 s versus 162 ± 27 s (P = 0.0186, paired Student's t test, n = 6; Fig.5D). Pumping pressures (P_pump) were essentially identical during nifedipine and placebo administration: 58 ± 3.1 mmHg and 58 ± 3.8 mmHg, respectively (P = 1.000, paired Student's t test, n = 6; Fig.5D). Packet velocity in the leg lymphatics was unaffected by nifedipine during inactivity or activity: during inactivity it was 13 ± 2 mm s⁻¹ and 12 ± 2 mm s⁻¹ with nifedipine or placebo administration, respectively (P = 0.4909, paired Student's t test, n = 6; Fig.5C). During activity the corresponding values were 12 ± 2 mm s⁻¹ and 11 ± 2 mm s⁻¹.

Figure 5. Nifedipine treatment affects lymphatic contractile function in vivo as measured with near-infrared fluorescence imaging.

A, cumulative data showing the contraction frequencies during inactivity and activity in the leg. A significantly increased frequency was observed during nifedipine treatment whereas this could not be confirmed during activity. B, corresponding values on the foot showed no statistical difference during inactivity or activity. C, packet velocity was only possible to measure on the leg and was unaffected by nifedipine. D, nifedipine treatment resulted in a significantly lower refill time whereas pumping pressures (P_pump) were unaffected. *P < 0.05 compared to placebo, paired Student's t test.

Foot

Contraction frequencies in lymphatic vessels in the foot did not differ between nifedipine and placebo treatments (Fig.5B): contraction frequencies during inactivity were 1.4 ± 0.2 min⁻¹ and 1.3 ± 0.3 min⁻¹ in the nifedipine and placebo periods, respectively (P = 0.7273, paired Student's t test, n = 6) corresponding values during activity were 1.8 ± 0.4 min⁻¹ and 2.0 ± 0.3 min⁻¹. Packet velocity was not measured because of the short distances and frequent branching pattern in the foot.

Discussion

Calcium channel blockers (CCB) have been in clinical use for treating hypertension since the 1970s. Early reports noted that peripheral oedema developed in many patients that received drugs from the DHP class (Bridgman, 1978; Guazzi et al. 1980; Husted et al. 1982). The oedema is considered to be a vasodilatory oedema (Messerli, 2001), caused by an increased capillary pressure leading to increased filtration, in accordance with the Starling principle (Starling, 1896). Whether fluid removal from the interstitial space is reduced due to DHP binding to the LSMC in addition to the ASMC has not been addressed. In this study we hypothesized that DHPs interfere with lymphatic contractile function and could contribute to oedema formation. We used a translational approach with focus on the human situation and started by investigating human lymphatic vessels in vitro and followed up these findings in vivo.

Nifedipine was our DHP of choice as it has been studied extensively and is the standard tool for investigating L-type calcium channel inhibition in vitro in addition to its clinical use. While nifedipine has been superseded by the similar drug amlodipine in modern hypertension treatment guidelines, the oedema incidence for the two drugs is the same. Amlodipine is, however, not practical for in vitro use because of the very long incubation times needed (Burges et al. 1987). Nifedipine and verapamil are 95% (van Bortel et al. 1989) and 90% (Keefe et al. 1981) bound to plasma proteins, respectively. Therapeutically relevant concentrations for nifedipine and verapamil are 2–90 ng ml⁻¹(Lederballe Pedersen et al. 1980; Kleinbloesem et al. 1987a; Akopov et al. 1994; Cainazzo et al. 2005) and 101–600 ng ml⁻¹ (Frishman et al. 1982), respectively, corresponding to free active plasma concentrations around 2–5 nmol l^–1 and 20–132 nmol l^–1. We tested the clinically relevant and achievable concentrations of these drugs for their ability to affect spontaneous contractions of lymphatic vessels in vitro. Verapamil required 10–50 times higher concentrations in vitro than that achieved in patients for blood pressure management and it is therefore unlikely that verapamil exerts a meaningful effect on the lymphatic vessels in vivo. This is consistent with clinical studies showing a low tendency of peripheral oedemas in patients administered verapamil (Anderson et al. 1999). The nifedipine concentrations required to abolish spontaneous contractions in vitro, however, corresponded to the clinically relevant free plasma concentrations of this DHP.

In vitro findings

When comparing the observed effect of nifedipine on lymphatic vessels with previous studies of arteries it was not possible to determine an IC₅₀ for nifedipine in LSMC, as changes in spontaneous activity were not graded but all-or-nothing. If one therefore compares the nifedipine concentration resulting in a complete cessation of phasic lymphatic contractions (pIC₁₀₀) with the nifedipine pIC₅₀ for relaxing human arteries (Sarsero et al. 1998) then the nifedipine IC₅₀ for arteries was ≥ to the IC₁₀₀ for lymphatic vessels despite the apparent expression of the relatively DHP-insensitive cardiac isoform of the Ca_v1.2 α1c subunit. The difference in sensitivity to nifedipine and verapamil was thus comparable or greater than that of human arteries (Schwinger et al. 1990; Sarsero et al. 1998). We also find that human lymphatic vessels are drastically more sensitive to DHP than animal vessels (Atchison & Johnston, 1997). Our results show that DHP sensitivity in human lymphatic vessels is essentially comparable to that in arteries and higher than in lymphatic vessels from animals. It is likely that lymphatic vessels in vivo are exposed to several vasoactive substances such as NA from perivascular nerves. It is therefore important to emphasize that not only spontaneous contractions but also NA-induced contractions in lymphatic vessels were markedly reduced or abolished by nifedipine. Nifedipine binds to the L-type VGCC in a voltage-dependent manner, with increased binding at more positive potentials; thus knowledge of the membrane potential is relevant to assess drug binding (Sanguinetti & Kass, 1984; Bean, 1984). Previous measurements of resting membrane potential in animal lymphatic vessels show that LSMCs (Ward et al. 1989; Van Helden, 1993; von der Weid & Van Helden, 1997) are 10–20 mV more hyperpolarized than arterial smooth muscle cells (ASMCs) (Jensen et al. 1993; Peng et al. 1998, 2001; Briggs Boedtkjer et al. 2013). We compared our wire myograph V_m measurements from lymphatic vessels with corresponding values from arteries, previously obtained in our lab, and found that LSMC V_m is comparable to ASMC V_m at around −45 mV. This supports an equivalent binding potential of nifedipine to both ASMC and LSMC. The wire myograph is a well-established technique for investigating contractile responses in a standardized manner. The absence of flow and thus shear stress and the lack of muscle shortening during a contraction are, however, significant factors that differ from the conditions of a lymphatic vessel in vivo.

In our database of thoracic duct experiments, including experiments for this study as well as other published and unpublished studies, we looked for a correlation between spontaneous activity in vitro and DHP treatment prior to surgery. We did not find any difference in spontaneous activity in vitro in patients treated with a DHP compared to patients not receiving DHP medication. The analysis included data generated from thoracic ducts maintained in a wire myograph from 126 patients, of which 27 were treated with a DHP. We recognize that this result will be affected to an unknown degree by the withdrawal of the patients’ medication a minimum of 12 h preoperatively. Furthermore, the tissue was transferred several times between solutions during transport and dissection and thus any residual DHP could have been washed out, thereby diminishing any effect the DHP may have had.

In our molecular analyses of L-type Ca_V we consistently detected the Ca_V1.2 isoform, as expected for smooth muscle, and we also observed other subtypes (Ca_V1.1 and 1.3), although less consistently. The presence of multiple subtypes is intriguing but may be attributable to the heterogeneous nature of the thoracic duct with interstitial cells (Briggs Boedtkjer et al. 2013) known to be present. Intriguingly, one could speculate that the variability in the concentration at which spontaneous activity was first affected by nifedipine may reflect this heterogeneous expression. The Ca_V1.2 gene contains an alternative splicing region that has an important influence on DHP sensitivity: exon 8 (smooth muscle cell isoform) or 8a (cardiac isoform) in the IS6 transmembrane domain. Inclusion of exon 8 imparts a 10-fold higher sensitivity to DHP than exon 8a (Zühlke et al. 1998). Unexpectedly, our PCR data show that the thoracic duct expresses exon 8a, which would be expected to lower the DHP sensitivity of Ca_V1.2, although exon8/exon8a mutants of Ca_V1.3 in a heterologous expression system have been observed to have similar DHP sensitivity (Huang et al. 2013). Furthermore, alternative splicing in the C-terminus drastically modulates DHP sensitivity in Ca_V1.2 (Zühlke et al. 1998) and Ca_V1.3 (Huang et al. 2013), so in spite of exon 8a inclusion the full-length LSMC Ca_V1.2 isoform may still be highly DHP sensitive. Future investigation of the relative expression of the different isoforms expressed, as well as the nature of the full-length transcripts and 1,4-dihydropyridine binding properties of the LSMC L-type VGCC protein, will help shed light on this apparent dichotomy.

Clinical trial: Lymphatic dysfunction as a cause of calcium channel blocker oedema

Our in vitro work convincingly demonstrates that nifedipine inhibits lymphatic pumping with equal potency in both small (≈300 μm internal diameter) and large (≈2 mm internal diameter) human lymphatic vessels. Previous studies have shown that nifedipine induces vasodilatation in blood vessels which leads to an increased fluid filtration, increased interstitial fluid pressure and in some cases ultimately oedema (Gustafsson, 1987; Pedrinelli et al. 2000, 2001). This perturbation of the interstitial space is normally compensated for by an increase in lymph flow (Aukland & Reed, 1993). The mechanism by which the increase in lymph flow occurs is likely to be an increase in both contraction frequency and contractility (Scallan et al. 2012). Administration of nifedipine to humans could therefore elicit two effects: (1) reduced lymphatic pumping, because of the inhibitory effect of nifedipine on the lymphatic vessels, or (2) increased lymphatic pumping because nifedipine increases the fluid filtration, leading to an increased load on the lymphatic vessels. We found both a lower refill time and increased contraction frequency when nifedipine was administered compared to placebo. An increased contraction frequency does not per se exclude an inhibitory effect of nifedipine on the lymphatic vessels, since the increase could have been even larger in a setting with hyperfiltration without nifedipine present. This suggests that the dominant effect of nifedipine in vivo in the study population is on the blood vasculature. With the other parameters measured we suggest that P_pump and packet velocity are the parameters that most closely describe the contractile state of the lymphatic vessels. The ability to generate systolic pressures and the velocity by which the lymph is propelled during a systole is presumably closely related to the contractility of the lymphangions. A reduced calcium influx due to L-type VGCC inhibition with nifedipine should be reflected by a reduction of these parameters; however, this was not observed. The P_pump estimate is only as sensitive as the pressure step incurred and the accuracy of the equipment controlling the pressure. With an average P_pump value of ≈60 mmHg, our 5 mmHg pressure steps (with ± 1 mmHg accuracy in the pressure level) would permit us to measure a ≈10% lowering of P_pump. Whether a greater sensitivity than this is required is unclear but it is doubtful whether such subtle changes are of physiological relevance. The discrepancy between the in vitro and in vivo results is perplexing. The in vitro results were consistent and obtained from a mixed group of 32 patients composed of both males and females with an age range of 30–80 years. Vessels harvested during cancer surgery came from a generally older population whereas the mesenteric lymphatics came from patients without malignancy and on average only 13 years older than the clinical trial population (39 years vs. 26 years). The in vitro findings were consistent and concentration dependent but our clinical trial measurements of the plasma concentration of nifedipine showed relevant concentrations that also were comparable to other clinical studies. We acknowledge that what is important for lymphatic function must be the concentration in the lymph and not in plasma but we did not determine this due to the technical demands and invasiveness needed to sample lymph. However, the observations that the concentration of other drugs in lymph (in animals) following systemic administration has been found to be comparable to plasma (Huang et al. 1991) and that nifedipine has a large tissue distribution (reflected by the quite large distribution volume of 0.8 l kg⁻¹; Kleinbloesem et al. 1984) indicate that nifedipine concentrations in lymph and plasma are similar. Extrapolating our in vitro observations from isolated lymphatic vessels directly to the in vivo situation is difficult as nifedipine also affects the arteries and fluid movement in the capillaries and thus also has an indirect effect on the lymphatics. An increased fluid filtration and load on the lymphatic vessels would presumably increase distension of the vessels, and the effectiveness of the nifedipine block during this condition was not investigated in vitro. To further investigate the discrepancy between the in vitro and in vivo results, it would therefore have been interesting to expose perfused vessels with low and high preload to nifedipine . Taken together, our in vitro and in vivo results demonstrate that VGCCs are important for lymphatic contractile function but when a VGCC blocker is administered systemically in vivo the dominant effect is that on the arteries.

New findings and perspective

In this study we present novel in vitro data on the importance of VGCCs for human lymphatic contractile function and a difference in sensitivity between DHPs and phenylalkamines. We furthermore show the necessity of VGCCs for generating action potentials in human lymphatic vessels. The clinical trial presented is the first of its kind to investigate the effect of a drug on the lymphatic circulation using NIRF imaging. NIRF imaging allows more detailed characterizations compared to lymphoscintigraphy and is the only method by which several dynamic parameters can be assessed quantitatively. Being the first drug intervention study, our findings also demonstrate the feasibility of NIRF imaging for assessing cardiovascular interventions on the lymphatic circulation. It is likely that many cardiovascular drugs also affect the lymphatic vasculature directly or indirectly and this area deserves more attention. Moreover, our study demonstrates that caution must be exercised when extrapolating results from isolated lymphatic vessels in vitro to the in vivo situation since, in the latter situation, there is an integrated effect on the arteries and veins. Thus our study highlights the importance of translational studies in lymphatic research.

Summary

We found convincing data in vitro showing that DHP inhibits lymphatic contractile function and electrical activity. These findings were in strong contrast to our in vivo results where we found an increased activity in the lymphatic vessels. Our in vivo findings suggest that the major effect of nifedipine in vivo is an increased fluid filtration leading to an increased activity in the lymphatic vessels. We cannot completely exclude the possibility that there is a DHP-induced inhibition of the lymphatic vessels that is overshadowed by the increased fluid filtration, but we could not find any direct evidence supporting this. We conclude that, in our study population of young healthy subjects, nifedipine affects the microcirculation by increasing the fluid filtration and that the lymphatic vasculature adapts to accommodate the increased fluid load.

Glossary

ASMC

arterial smooth muscle cell

CCB

calcium channel blocker

CCRC

cumulative concentration–response curve

DHP

dihydropyridine

LSMC

lymphatic smooth muscle cell

ML

mesenteric lymphatic

NA

noradrenaline

NIRF

near-infrared fluorescence

P_pump

pumping pressure

RMP

resting membrane potential

TD

thoracic duct

VGCC

voltage-gated calcium channel

V_m

membrane potential

Key points

• We studied the effects of antihypertensive calcium channel blockers on Ca_V1.2, 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.

Additional information

Competing interests

None declared.

Author contributions

In vitro experiments were conducted in Aalkjaer lab at the Department of Biomedicine, Aarhus University. N.T., C.A., D.B. and V.H. conceived the project and interpreted data. H.P., E.P. and J.N. provided tissue. N.T. and D.B. designed the experiments and analysed the data. N.T., J.M., S.M., D.B. and S.K. collected data. The clinical trial was conducted at the Department of Cardiothoracic surgery, Aarhus University Hospital, by N.T. N.T. and D.B. wrote the first draft of the manuscript. All authors revised the manuscript and approved the final version.

Funding

This work was supported by the Oak Foundation, the Water and Salt Research Centre (Danish Research Council), Erik and Knudsine Leijons Mindegave, Agnes Niebuhr Andersson's Cancer Research Fund and Aarhus University Hospital.

Translational perspective

Calcium channel blockers are one of the most widely prescribed anti-hypertension therapies; unfortunately 10–30% of those undergoing treatment with these drugs, in particular dihydropyridines, develop peripheral oedema as a consequence of treatment. An off-target effect on the smooth muscle cells of lymphatic vessels that would reduce removal of interstitial fluid has not yet been studied in the pathogenesis of dihydropyridine-related oedema formation. We found that human lymphatic vessels were sensitive to therapeutic concentrations of dihydropyridines in vitro, which caused spontaneous contractions to stop. However in vivo we found no similar evidence for an inhibition of lymphatic pumping. The in vivo data comes from the first randomized clinical trial investigating the effect of a drug on the lymphatic circulation where the pre-defined end-points are parameters that reflect lymphatic function. Being the first drug intervention study, our findings also demonstrate the feasibility of using near-infrared fluorescence imaging for assessing cardiovascular interventions on the lymphatic circulation. It is likely that many cardiovascular drugs also affect the lymphatic vasculature directly or indirectly and this area deserves more attention. Moreover, our study demonstrates that care must be taken when extrapolating results from isolated lymphatic vessels in vitro to in vivo. Thus our study highlights the importance of translational studies in lymphatic research.

Supporting Information

The following supporting information is available in the online version of this article.

Movie S1

Video demonstrating NIRF imaging of lymphatic vessels on the foot. The video is speeded up x3.

Movie S2

Video demonstrating NIRF imaging of lymphatic vessels on the leg. The video is speeded up x3.

Movie S3

Video demonstrating a refill time sequence. The video is speeded up x3.

Movie S4

Video showing how lymph crosses an inflatable cuff when the pressure is lowered to 60 mm Hg. The video is speeded up x3.

Photograph S1

Photograph of the area shown in movie S1

Photograph S2

Photograph of the area shown in movie S2

Photograph S3

Photograph of the area shown in movie S3