Real-Time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels.

The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i). This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i and diameter measurements continuously in real time. The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Drug-Related Lymphedema: Mysteries, Mechanisms, and Potential Therapies.

The lymphatic circulation is an important component of the circulatory system in humans, playing a critical role in the transport of lymph fluid containing proteins, white blood cells, and lipids from the interstitial space to the central venous circulation. The efficient transport of lymph fluid critically relies on the rhythmic contractions of collecting lymph vessels, which function to "pump" fluid in the distal to proximal direction through the lymphatic circulation with backflow prevented by the presence of valves. When rhythmic contractions are disrupted or valves are incompetent, the loss of lymph flow results in fluid accumulation in the interstitial space and the development of lymphedema. There is growing recognition that many pharmacological agents modify the activity of ion channels and other protein structures in lymph muscle cells to disrupt the cyclic contraction and relaxation of lymph vessels, thereby compromising lymph flow and predisposing to the development of lymphedema. The effects of different medications on lymph flow can be understood by appreciating the intricate intracellular calcium signaling that underlies the contraction and relaxation cycle of collecting lymph vessels. For example, voltage-sensitive calcium influx through long-lasting ("L-type") calcium channels mediates the rise in cytosolic calcium concentration that triggers lymph vessel contraction. Accordingly, calcium channel antagonists that are mainstay cardiovascular medications, attenuate the cyclic influx of calcium through L-type calcium channels in lymph muscle cells, thereby disrupting rhythmic contractions and compromising lymph flow. Many other classes of medications also may contribute to the formation of lymphedema by impairing lymph flow as an off-target effect. The purpose of this review is to evaluate the evidence regarding potential mechanisms of drug-related lymphedema with an emphasis on common medications administered to treat cardiovascular diseases, metabolic disorders, and cancer. Additionally, although current pharmacological approaches used to alleviate lymphedema are largely ineffective, efforts are mounting to arrive at a deeper understanding of mechanisms that regulate lymph flow as a strategy to identify novel anti-lymphedema medications. Accordingly, this review also will provide information on studies that have explored possible anti-lymphedema therapeutics.

Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation.

Background and Purpose: Doxorubicin (DOX) is a risk factor for arm lymphedema in breast cancer patients. We reported that DOX opens ryanodine receptors (RYRs) to enact "calcium leak," which disrupts the rhythmic contractions of lymph vessels (LVs) to attenuate lymph flow. Here, we evaluated whether dantrolene, a clinically available RYR1 subtype antagonist, prevents the detrimental effects of DOX on lymphatic function. Experimental Approach: Isolated rat mesenteric LVs were cannulated, pressurized (4-5 mm Hg) and equilibrated in physiological salt solution and Fura-2AM. Video microscopy recorded changes in diameter and Fura-2AM fluorescence tracked cytosolic free calcium ([Ca2+ i]). High-speed in vivo microscopy assessed mesenteric lymph flow in anesthetized rats. Flow cytometry evaluated RYR1 expression in freshly isolated mesenteric lymphatic muscle cells (LMCs). Key Results: DOX (10 µmol/L) increased resting [Ca2+ i] by 17.5 ± 3.7% in isolated LVs (n = 11). The rise in [Ca2+ i] was prevented by dantrolene (3 µmol/L; n = 10). A single rapid infusion of DOX (10 mg/kg i.v.) reduced positive volumetric lymph flow to 29.7 ± 10.8% (n = 7) of baseline in mesenteric LVs in vivo. In contrast, flow in LVs superfused with dantrolene (10 µmol/L) only decreased to 76.3 ± 14.0% (n = 7) of baseline in response to DOX infusion. Subsequently, expression of the RYR1 subtype protein as the presumed dantrolene binding site was confirm in isolated mesenteric LMCs by flow cytometry. Conclusion and Implications: We conclude that dantrolene attenuates the acute impairment of lymph flow by DOX and suggest that its prophylactic use in patients subjected to DOX chemotherapy may lower lymphedema risk.

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 STATEMENT: ATP-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.

Calcium Channel Blockers and Risk of Lymphedema among Breast Cancer Patients: Nested Case-Control Study.

BACKGROUND: To assess the risk of lymphedema associated with the use of calcium channel blockers (CCB) among breast cancer patients. METHODS: A nested case-control study of adult female breast cancer patients receiving an antihypertensive agent was conducted using administrative claims data between 2007 and 2015. Cases were patients with lymphedema who were matched to 5 controls based on nest entry date (±180 days), age (±5 years), number of hypertensive drug classes, Charlson Comorbidity Index (CCI), thiazide exposure, and insurance type. Exposure to CCBs and covariates was identified in the 180-day period prior to event date. Conditional logistic regression was used to assess the impact of exposure among cases and controls. RESULTS: A total of 717 cases and 1,681 matched controls were identified. After matching on baseline characteristics, mastectomy (7.8% vs. 4.8%; P = 0.0039), exposure to radiotherapy (27.1% vs. 21.7%; P = 0.0046), taxane-based chemotherapy (11.7% vs. 7.4%; P = 0.0007), anthracycline-based chemotherapy (6.0% vs. 3.6%; P = 0.0073), CCB use (28.3% vs. 23.3%; P = 0.0087), and CCI (19.8% vs. 12.7%; P < 0.0001; score of 4 or above) were all higher in cases during the 180 days prior to the event date. In the adjusted analysis, CCB exposure was significantly associated with increased risk of lymphedema (OR = 1.320; 95% confidence interval, 1.003-1.737). CONCLUSIONS: CCB use was significantly associated with the development of lymphedema in breast cancer patients. IMPACT: CCBs should be avoided or used with caution in breast cancer patients to reduce the risk for developing lymphedema.

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 µmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 µmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

High-speed microscopy for in vivo monitoring of lymph dynamics.

The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels (LVs) and automated quantification of lymphatic function at a temporal resolution of 2 milliseconds. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the LVs were tracked to estimate flow velocity. The system performance initially was verified in vitro using 6- and 10-µm microspheres as cell phantoms on slides and in 90-µm diameter tubes at flow velocities up to 4 cm/second. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of the mesentery. The system revealed reductions of mesenteric LV contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during lymphatic system diseases.

Fenofibrate Attenuates Neutrophilic Inflammation in Airway Epithelia: Potential Drug Repurposing for Cystic Fibrosis.

A hallmark of cystic fibrosis (CF) lung disease is neutrophilic airway inflammation. Elevated neutrophil counts have been associated with decreased forced expiratory volume in 1 second and poor clinical measures in patients with CF. Interleukin 8 (IL-8), epithelial neutrophil activating protein 78 (ENA-78), tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) contribute to neutrophil activation and disease pathogenesis in the airways of patients with CF. Drugs that modify the production of these chemokines in the airways could potentially benefit CF patients. Thus, we determined the effects of fenofibrate on their production in cell populations obtained from the airways. Human small airway epithelial cells and CF bronchial epithelial cells were treated with IL-1ß to induce inflammation. We cotreated the cells with fenofibrate at concentrations ranging from 10 to 50 µM to determine if this drug could attenuate the inflammation. IL-8, ENA-78, TNF-α, GM-CSF, and G-CSF production were measured from the cell culture supernates by ELISA. ANOVA statistical testing was conducted using SPSS 17.0. IL-1ß increased the production of each of the chemokines by several fold. Fenofibrate reduced IL-1ß induced production of each of these neutrophilic chemokines at the concentrations used. IL-1ß increases the production of neutrophilic chemokines in airway epithelial cells. Cotreatment with fenofibrate blunts these processes. Fenofibrate should be explored as a therapeutic option to modulate the abundant neutrophilic inflammation observed in CF.

Gender Differences in Cardiovascular Drugs.

The different responses of women and men to cardiovascular drugs reflect gender -specific variances in pharmacokinetic profiles and drug sensitivities coupled to inherent differences in the underlying physiology of each sex. Thus, many common cardiovascular drugs exhibit gender -specific therapeutic and adverse effects. For example, the QT interval of the electrocardiogram is longer in women compared to men, and accordingly, drugs that prolong the QT interval are more likely to cause lethal ventricular arrhythmias in female than male patients. As more clinical drug trials include women subjects, our improved knowledge base for assessing the risk/benefit ratio for cardiovascular drugs in women will enable us to consider gender as one factor in prescribing drugs and adjusting drug loading and maintenance dosages. This short review will present evidence for gender- related differences in the responses to common cardiovascular drugs including statins, antiplatelet and antithrombotic agents, ß-blockers, digoxin, vasodilator therapies, and drugs associated with the Long QT Syndrome.