A combination of extrinsic (passive) and intrinsic (active) forces move lymph against a hydrostatic pressure gradient in most regions of the body. The effectiveness of the lymph pump system impacts not only interstitial fluid balance but other aspects of overall homeostasis. This review focuses on the mechanisms that regulate the intrinsic, active contractions of collecting lymphatic vessels in relation to their ability to actively transport lymph. Lymph propulsion requires not only robust contractions of lymphatic muscle cells, but contraction waves that are synchronized over the length of a lymphangion as well as properly functioning intraluminal valves. Normal lymphatic pump function is determined by the intrinsic properties of lymphatic muscle and the regulation of pumping by lymphatic preload, afterload, spontaneous contraction rate, contractility and neural influences. Lymphatic contractile dysfunction, barrier dysfunction and valve defects are common themes among pathologies that directly involve the lymphatic system, such as inherited and acquired forms of lymphoedema, and pathologies that indirectly involve the lymphatic system, such as inflammation, obesity and metabolic syndrome, and inflammatory bowel disease.

We identified a regional dichotomy in murine lymphatic contractile function with regard to vessel location within the periphery or visceral cavity. All vessels isolated from peripheral regions [cervical, popliteal, inguinal, axillary, and internodal inguinal axillary (Ing-Ax)] developed robust contractions with maximal ejection fractions (EFs) of 50-80% in our ex vivo isobaric myograph experiments. Conversely, vessels isolated from the visceral cavity (mesenteric, thoracic duct, and iliac) demonstrated maximal EFs of ≤10%. Using pressure myography, sharp electrode membrane potential recordings, and Ca imaging, we assessed the role of L-type Ca channels in this contractile dichotomy. Ing-Ax membrane potential revealed a ~2-s action potential (AP) cycle (resting -35 mV, spike -5 mV, and plateau -11 mV) with a plateau phase that was significantly lengthened by the L-type Ca channel agonist Bay K8644 (BayK; 100 nM). APs recorded from mesenteric vessels, however, displayed a slower upstroke and an elongated time over threshold. BayK (100 nM) increased the mesenteric AP upstroke velocity and plateau duration but also significantly hyperpolarized the vessel. Contractions of vessels from both regions were preceded by Ca flashes, detected with a smooth muscle-specific endogenous Ca reporter, that typically were coordinated over the length of the vessel. Similar to the membrane potential recordings, Ca flashes in mesenteric vessels were weaker and had a slower rise time but were longer lasting than those in Ing-Ax vessels. BayK (100 nM) significantly increased the Ca transient amplitude and duration in both vessels and decreased time to peak Ca in mesenteric vessels. However, a higher concentration (1 μM) of BayK was required to produce even a modest increase in EF in visceral lymphatics, which remained at <20%. NEW & NOTEWORTHY Lymphatic collecting vessels isolated from murine peripheral tissues, but not from the visceral cavities, display robust contractile behavior similar to lymphatic vessels from other animal models and humans. These differences are partially explained by L-type Ca channel activity as revealed by the first measurements of murine lymphatic action potentials and contraction-associated Ca transients.

Biocatalytic electrodes made of buckypaper were modified with PQQ‐dependent glucose dehydrogenase on the anode and with laccase on the cathode. The enzyme modified electrodes were assembled in a biofuel cell which was first characterized in human serum solution and then the electrodes were placed onto exposed rat cremaster tissue. Glucose and oxygen dissolved in blood were used as the fuel and oxidizer, respectively, for the implanted biofuel cell operation. The steady‐state open circuitry voltage of 140±30 mV and short circuitry current of 10±3 µA (current density ca. 5 µA cm−2 based on the geometrical electrode area of 2 cm2) were achieved in the in vivo operating biofuel cell. Future applications of implanted biofuel cells for powering of biomedical and sensor devices are discussed.

Rationale: Mutations in GJC2 and GJA1, encoding connexins (Cxs) 47 and 43 respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and the electrical coupling of those through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels. Objective: Determine which Cx isoforms in LMCs and/or lymphatic endothelial cells (LECs) are required for the entrainment of lymphatic contraction waves and efficient lymph transport. Methods and Results: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and LEC layers. Genetic deletion of the major LEC Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle-specific Cx45 deficiency resulted in 10–18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45-deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load. Conclusions: Our results: 1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; 2) show that major endothelial Cxs are dispensable for the entrainment of contractions; 3) reveal a lack of coupling between LECs and LMCs, in contrast to arterioles; 4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and 5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.

, is an independent predictor of cardiovascular event risk. Recent evidence demonstrates that accelerated aortic stiffening occurs in obesity; however, little is known regarding stiffening of other disease-relevant arteries or whether regional variation in arterial stiffening occurs in this setting. We addressed this gap in knowledge by assessing femoral PWV in vivo in conjunction with ex vivo analyses of femoral and coronary structure and function in a mouse model of Western diet (WD; high-fat/high-sugar)-induced obesity and insulin resistance. WD feeding resulted in increased femoral PWV in vivo. Ex vivo analysis of femoral arteries revealed a leftward shift in the strain-stress relationship, increased modulus of elasticity, and decreased compliance indicative of increased stiffness following WD feeding. Confocal and multiphoton fluorescence microscopy revealed increased femoral stiffness involving decreased elastin/collagen ratio in conjunction with increased femoral transforming growth factor-␤ (TGF-␤) content in WD-fed mice. Further analysis of the femoral internal elastic lamina (IEL) revealed a significant reduction in the number and size of fenestrae with WD feeding. Coronary artery stiffness and structure was unchanged by WD feeding. Functionally, femoral, but not coronary, arteries exhibited endothelial dysfunction, whereas coronary arteries exhibited increased vasoconstrictor responsiveness not present in femoral arteries. Taken together, our data highlight important regional variations in the development of arterial stiffness and dysfunction associated with WD feeding. Furthermore, our results suggest TGF-␤ signaling and IEL fenestrae remodeling as potential contributors to femoral artery stiffening in obesity.

the spontaneous contractions of collecting lymphatic vessels provide an essential propulsive force to return lymph centrally. these contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. in previous studies, t-type voltage-gated ca 2+ channels (VGccs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and ni 2+. Our goal was to test this idea in a more definitive way using genetic knock out mice. first, we demonstrated through both pcR and immunostaining that mouse lymphatic muscle cells expressed ca v 3.1 and Ca v 3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each t-type VGcc isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied ex vivo. further, both Wt and ca v 3.1 −/− ; 3.2 −/− double knockout lymphatic vessels responded similarly to mibefradil and ni 2+ , which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than t-type VGccs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and ca v 3.1 −/− ; 3.2 −/− double knockout mice. in contrast, smoothmuscle specific deletion of the L-type VGCC, Ca v 1.2, completely abolished all lymphatic spontaneous contractions. collectively our results suggest that, although t-type VGccs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and ni 2+ in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions. The spontaneous contractions of collecting lymphatic vessels propel lymph centrally to account for 2/3 of peripheral lymph flow 1,2. These rapid, large-amplitude contractions are analogous to twitch contractions of cardiac and skeletal muscle and are particularly important for moving lymph uphill against the adverse hydrostatic gradients that exist in dependent extremities. Lymphatic contractions are triggered by action potentials (APs) in lymphatic smooth muscle cell...

The lymphatic system plays a key role in tissue fluid homeostasis, immune cell trafficking, and fat absorption. We previously reported a bacterial artificial chromosome (BAC)-based lymphatic reporter mouse, where EGFP is expressed under the regulation of the Prox1 promoter. This reporter line has been widely used to conveniently visualize lymphatic vessels and other Prox1-expressing tissues such as Schlemm’s canal. However, mice have a number of experimental limitations due to small body size. By comparison, laboratory rats are larger in size and more closely model the metabolic, physiological, and surgical aspects of humans. Here, we report development of a novel lymphatic reporter rat using the mouse Prox1-EGFP BAC. Despite the species mismatch, the mouse Prox1-EGFP BAC enabled a reliable expression of EGFP in Prox1-expressing cells of the transgenic rats and allowed a convenient visualization of all lymphatic vessels, including those in the central nervous system, and Schlemm’s canal. To demonstrate the utility of this new reporter rat, we studied the contractile properties and valvular functions of mesenteric lymphatics, developed a surgical model for vascularized lymph node transplantation, and confirmed Prox1 expression in venous valves. Together, Prox1-EGFP rat model will contribute to the advancement of lymphatic research as a valuable experimental resource.

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