Cancer-related lymphedema is an accumulation of protein-rich fluid in the interstitial tissue, which results from compromised lymphatic drainage associated with cancer treatments involving radiation and/or surgical removal of lymph nodes. Once established, lymphedema is irreversible, and there is no targeted pharmacological therapy. This dissertation project focuses on an unrecognized mechanism by which the most commonly used anthracycline chemotherapeutic drug, doxorubicin (DOX), may contribute to the development of lymphedema.
The primary "mechanical" role of the lymphatic vasculature is fluid homeostasis, which is achieved through spontaneous rhythmic contractions of lymph vessels (LV) that propel fluid from the extracellular space to the central venous ducts and prevent excess fluid accumulation in tissues. Accordingly, studies of this dissertation project characterize the effect of DOX on lymphatic contractile function and lymph flow. Initial studies were conducted using isolated, cannulated LVs from the rat mesentery and contractile function was measured using real-time edge detection software to track vessel diameter. These studies analyzed changes in LV contractile behavior in response to clinically relevant plasma concentrations of DOX. Measurements of contractile parameters included LV basal diameter, contraction amplitude and frequency, and integrated contraction response (area under the curve (AUC)). DOX significantly decreased all four contractile parameters when compared to baseline and produced a tonic constriction of LVs characterized by loss of rhythmic contractions. These effects were partially reversed by washout of DOX with drug-free solution, suggesting the effect of DOX on LV contractions is separate from its cytotoxic actions.
Lymph fluid contains an array of components, such as proteins, lipids, and immune cells that may influence LV contractile function, which are not present in isolated LVs. In an anesthetized rat, we placed an intestinal loop on a heated microscope stage equipped with a recirculating bath that enabled us to superfuse defined DOX concentrations across in situ mesenteric LVs. Customized cell tracking software was used to measure lymph flow in high speed video recordings of LVs before and after DOX superfusion. Continuous superfusion of DOX progressively decreased positive cell velocity and positive volumetric flow, suggesting that in situ LVs are more sensitive to DOX inhibition compared to isolated LVs.
Our ultimate goal was to measure lymph flow in vivo after IV administration of a clinically relevant dose of DOX. In order to accomplish this goal, it was necessary to identify a time interval during which we could measure the effect of clinically relevant plasma concentrations of DOX on lymph flow in vivo. Accordingly, pharmacokinetic studies were conducted to determine elimination phases for DOX after IV administration via tail vein. DOX is known to exhibit biphasic elimination after IV injection, characterized by an initial rapid phase that reflects the rapid redistribution of DOX from blood to tissues, followed by a slower plateau phase caused by tissue elimination of DOX, presumably involving lymphatic uptake. It was our goal to identify the start of this slow plateau phase after IV injection of DOX as a surrogate indicator of initial DOX entry into the lymphatic vasculature. Thus, the studies of Specific Aim 1 provided strong initial evidence that DOX acutely and directly suppresses lymphatic contractile function and lymph flow.
Subsequently, studies of this dissertation project were designed to evaluate the therapeutic potential of a clinically-available RyR1 blocker, dantrolene (DANT), to prevent the disruption of lymphatic contractions by DOX. Initial immunocytochemical studies sought to determine whether the RyR1 isoform is expressed by isolated rat mesenteric LSMCs. LSMCs were incubated with anti-RyR1 primary antibody and Cy3-conjugated anti-rabbit secondary antibody, and fluorescent signal was detected using confocal microscopy. Anti-RyR1 immunofluorescence was detected in isolated LSMCs and also in skeletal muscle cross sections, which were used as a positive control for RyR1 expression. Next we determined whether RyR blockade by a clinically-relevant concentration of DANT inhibited lymphatic contractile function in isolated LVs. DANT did not affect basal LV contractile parameters.
In conclusion, DOX at clinically-relevant concentrations acutely and directly suppresses LV contractile function and lymph flow, which potentially contributes to the development of lymphedema. The inhibitory effect of DOX on the rhythmic contractions of isolated rat mesenteric LVs is reversed by drug washout, suggesting this inhibition is distinct from the cytotoxic action of DOX. DOX exerted similar inhibitory effects on LV contraction as ryanodine, a pharmacological activator of RyRs. Thus, our findings suggest that DOX acutely inhibits contractile function in rat mesenteric LVs by activating RyRs in LSMCs. Also DOX-induced inhibition of lymphatic contractile function was partially prevented by DANT, a clinically available RyR1 blocker. Dantrolene, a drug already approved for the clinical treatment of muscle spasm and malignant hyperthermia, may represent a potential anti-lymphedema therapeutic to prevent the off-target effect of DOX on lymphatic contractile function. (Abstract shortened by ProQuest.)
|Advisor:||Rusch, Nancy J.|
|Commitee:||Galanzha, Ekaterina, Klimberg, Suzanne, Light, Kim E., Mu, Shengyu, Rhee, Sung W.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 78/02(E), Dissertation Abstracts International|
|Keywords:||Doxorubicin, Lymph vessel, Lymphatic function, Lymphatic vessel, Lymphedema, Ryanodine receptor|
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