Dichotomous effects of in vivo and in vitro ionizing radiation exposure on lymphatic function.

On the one hand, lymphatic dysfunction induces interstitial edema and inflammation. On the other hand, the formation of edema and inflammation induce lymphatic dysfunction. However, informed by the earlier reports of undetected apoptosis of irradiated lymphatic endothelial cells (LECs) in vivo, lymphatic vessels are commonly considered inconsequential to ionizing radiation (IR)-induced inflammatory injury to normal tissues. Primarily because of the lack of understanding of the acute effects of IR exposure on lymphatic function, acute edema and inflammation, common sequelae of IR exposure, have been ascribed solely to blood vessel damage. Therefore, in the present study, the lymphatic acute responses to IR exposure were quantified to evaluate the hypothesis that IR exposure impairs lymphatic pumping. Rat mesenteric lymphatic vessels were irradiated in vivo or in vitro, and changes in pumping were quantified in isolated vessels in vitro. Compared with sham-treated vessels, pumping was lowered in lymphatic vessels irradiated in vivo but increased in vessels irradiated in vitro. Furthermore, unlike in blood vessels, the acute effects of IR exposure in lymphatic vessels were not mediated by nitric oxide-dependent pathways in either in vivo or in vitro irradiated vessels. After cyclooxygenase blockade, pumping was partially restored in lymphatic vessels irradiated in vitro but not in vessels irradiated in vivo. Taken together, these findings demonstrated that lymphatic vessels are radiosensitive and LEC apoptosis alone may not account for all the effects of IR exposure on the lymphatic system.NEW & NOTEWORTHY Earlier studies leading to the common belief that lymphatic vessels are radioresistant either did not characterize lymphatic pumping, deemed necessary for the resolution of edema and inflammation, or did it in vivo. By characterizing pumping in vitro, the present study, for the first time, demonstrated that lymphatic pumping was impaired in vessels irradiated in vivo and enhanced in vessels irradiated in vitro. Furthermore, the pathways implicated in ionizing radiation-induced blood vessel damage did not mediate lymphatic responses.

Analysis of Lymphatic Vessel Formation by Whole-Mount Immunofluorescence Staining.

Pathological alterations of lymphatic structure and function interfere with lymph transport, resulting in a wide range of clinical disorders that include edema, tissue inflammation, and metabolic syndromes. Mesentery contains abundant lymphatic vessels and plays an important role in transporting absorbed lipid from the intestine. In this manuscript, we describe a whole-mount staining method on isolated mouse mesentery with VEGFR3, Prox1, and Lyve1 antibodies to visualize the morphology of lymphatic vessels.

Inflammatory state of lymphatic vessels and miRNA profiles associated with relapse in ovarian cancer patients.

Lymphogenic spread is associated with poor prognosis in epithelial ovarian cancer (EOC), yet little is known regarding roles of non-peri-tumoural lymphatic vessels (LVs) outside the tumour microenvironment that may impact relapse. The aim of this feasibility study was to assess whether inflammatory status of the LVs and/or changes in the miRNA profile of the LVs have potential prognostic and predictive value for overall outcome and risk of relapse. Samples of macroscopically normal human lymph LVs (n = 10) were isolated from the external iliac vessels draining the pelvic region of patients undergoing debulking surgery. This was followed by quantification of the inflammatory state (low, medium and high) and presence of cancer-infiltration of each LV using immunohistochemistry. LV miRNA expression profiling was also performed, and analysed in the context of high versus low inflammation, and cancer-infiltrated versus non-cancer-infiltrated. Results were correlated with clinical outcome data including relapse with an average follow-up time of 13.3 months. The presence of a high degree of inflammation correlated significantly with patient relapse (p = 0.033). Cancer-infiltrated LVs showed a moderate but non-significant association with relapse (p = 0.07). Differential miRNA profiles were identified in cancer-infiltrated LVs and those with high versus low inflammation. In particular, several members of the let-7 family were consistently down-regulated in highly inflamed LVs (>1.8-fold, p<0.05) compared to the less inflamed ones. Down-regulation of the let-7 family appears to be associated with inflammation, but whether inflammation contributes to or is an effect of cancer-infiltration requires further investigation.

IL-1ß reduces cardiac lymphatic muscle contraction via COX-2 and PGE2 induction: Potential role in myocarditis.

The role of lymphatic vessels in myocarditis is largely unknown, while it has been shown to play a key role in other inflammatory diseases. We aimed to investigate the role of lymphatic vessels in myocarditis using in vivo model induced with Theiler's murine encephalomyelitis virus (TMEV) and in vitro model with rat cardiac lymphatic muscle cells (RCLMC). In the TMEV model, we found that upregulation of a set of inflammatory mediator genes, including interleukin (IL)-1ß, tumor necrosis factor (TNF)-αand COX-2 were associated with disease activity. Thus, using in vitro collagen gel contraction assays, we decided to clarify the role(s) of these mediators by testing contractility of RCLMC in response to IL-1ß and TNF-α individually and in combination, in the presence or absence of: IL-1 receptor antagonist (Anakinra); cyclooxygenase (COX) inhibitors inhibitors (TFAP, diclofenac and DuP-697). IL-1ß impaired RCLMC contractility dose-dependently, while co-incubation with both IL-1ß and TNF-α exhibited synergistic effects in decreasing RCLMC contractility with increased COX-2 expression. Anakinra maintained RCLMC contractility; Anakinra blocked the mobilization of COX-2 induced by IL-1ß with or without TNF-α. COX-2 inhibition blocked the IL-1ß-mediated decrease in RCLMC contractility. Mechanistically, we found that IL-1ß increased prostaglandin (PG) E2 release dose-dependently, while Anakinra blocked IL-1ß mediated PGE2 release. Using prostaglandin E receptor 4 (EP4) receptor antagonist, we demonstrated that EP4 receptor blockade maintained RCLMC contractility following IL-1ß exposure. Our results indicate that IL-1ß reduces RCLMC contractility via COX-2/PGE2 signaling with synergistic cooperation by TNF-α. These pathways may help provoke inflammatory mediator accumulation within the heart, driving progression from acute myocarditis into dilated cardiomyopathy.

Enhancing Renal Lymphatic Expansion Prevents Hypertension in Mice.

RATIONALE: Hypertension is associated with renal infiltration of activated immune cells; however, the role of renal lymphatics and immune cell exfiltration is unknown. OBJECTIVE: We tested the hypotheses that increased renal lymphatic density is associated with 2 different forms of hypertension in mice and that further augmenting renal lymphatic vessel expansion prevents hypertension by reducing renal immune cell accumulation. METHODS AND RESULTS: Mice with salt-sensitive hypertension or nitric oxide synthase inhibition-induced hypertension exhibited significant increases in renal lymphatic vessel density and immune cell infiltration associated with inflammation. Genetic induction of enhanced lymphangiogenesis only in the kidney, however, reduced renal immune cell accumulation and prevented hypertension. CONCLUSIONS: These data demonstrate that renal lymphatics play a key role in immune cell trafficking in the kidney and blood pressure regulation in hypertension.

Citrus nomilin down-regulates TNF-α-induced proliferation of aortic smooth muscle cells via apoptosis and inhibition of IκB.

Nomilin is a bitter compound present in citrus and has been demonstrated as useful for various disease preventions through anti-proliferative, anti-inflammatory, and pro-apoptotic activities. Although in vitro disease models have shown that certain limonoids in the p38 mitogen-activated protein kinase signal cascade, the downstream signaling pathways remain unclear. In this study, the effects of nomilin on the proliferation and apoptotic pathways of human aortic smooth muscle cells (HASMCs) that forms the basis of progression of atherosclerotic diseases and restenosis was tested for the first time. The cellular uptake level and stability of nomilin were determined by high-performance liquid chromatography and high-resolution mass spectra. Pretreatment of HASMCs with nomilin stimulated extrinsic caspase-8, intrinsic caspase-9, and apoptotic caspase-3 and resulted in significant inhibition of TNF-α-induced proliferation. Additionally, results showed a decreased ratio of anti-apoptotic Bcl-2 protein to pro-apoptotic Bax (Bcl2/Bax), indicating mitochondrial dysfunction consistent with apoptosis. Furthermore, nomilin significantly decreased the phosphorylation of IκBα, an inhibitor of NF-κB and subsequently, reduced the downstream inflammatory signaling in TNF-α treated HASMCs. Our findings indicate that the anti-proliferative activity of nomilin on TNF-α-induced HASMCs results from apoptosis through a mitochondrial-dependent pathway and suppression of inflammatory signaling mediated through NF-κB.

Macrophage alterations within the mesenteric lymphatic tissue are associated with impairment of lymphatic pump in metabolic syndrome.

OBJECTIVE: The intrinsic lymphatic pump is critical to proper lymph transport and is impaired in models of the MetSyn. Lymphatic contractile inhibition under inflammatory conditions has been linked with elevated NO production by activated myeloid-derived cells. Hence we hypothesized that inhibition of the MLV pump function in MetSyn animals was dependent on NO and was associated with altered macrophage recruitment and polarization within the MLV. METHODS: We used a high fructose-fed rat model of MetSyn. Macrophage polarization was determined by whole mount immunofluorescence in mesenteric neurovascular bundles based on expression of CD163, CD206, and MHCII. We also utilized isolated vessel isobaric preparations to determine the role for elevated NO production in the inhibition of MLV contractility. Both LECs and LMCs were used to assess the cytokines and chemokines to test how the lymphatic cells response to inflammatory conditions. RESULTS: Data demonstrated a greater accumulation of M1-skewed (CD163+ MHCII+ ) macrophages that were observed both within the perivascular adipose tissue and invested along the lymphatic vessels in MetSyn rats when compared to control rats. LECs and LMCs basally express the macrophage maturation polarization cytokines monocyte colony-stimulating factor and dramatically up regulate the M1 promoting cytokine granulocyte/monocyte colony-stimulating factor in response to lipopolysaccharide stimulation. MetSyn MLVs exhibited altered phasic contraction frequency. Incubation of MetSyn MLVs with LNAME or Glib had a partial restoration of lymphatic contraction frequency. CONCLUSION: The data presented here provide the first evidence for a correlation between alterations in macrophage status and lymphatic dysfunction that is partially mediated by NO and KATP channel in MetSyn rats.

Lipopolysaccharide modulates neutrophil recruitment and macrophage polarization on lymphatic vessels and impairs lymphatic function in rat mesentery.

Impairment of the lymphatic system is apparent in multiple inflammatory pathologies connected to elevated endotoxins such as LPS. However, the direct mechanisms by which LPS influences the lymphatic contractility are not well understood. We hypothesized that a dynamic modulation of innate immune cell populations in mesentery under inflammatory conditions perturbs tissue cytokine/chemokine homeostasis and subsequently influences lymphatic function. We used rats that were intraperitoneally injected with LPS (10 mg/kg) to determine the changes in the profiles of innate immune cells in the mesentery and in the stretch-mediated contractile responses of isolated lymphatic preparations. Results demonstrated a reduction in the phasic contractile activity of mesenteric lymphatic vessels from LPS-injected rats and a severe impairment of lymphatic pump function and flow. There was a significant reduction in the number of neutrophils and an increase in monocytes/macrophages present on the lymphatic vessels and in the clear mesentery of the LPS group. This population of monocytes and macrophages established a robust M2 phenotype, with the majority showing high expression of CD163 and CD206. Several cytokines and chemoattractants for neutrophils and macrophages were significantly changed in the mesentery of LPS-injected rats. Treatment of lymphatic muscle cells (LMCs) with LPS showed significant changes in the expression of adhesion molecules, VCAM1, ICAM1, CXCR2, and galectin-9. LPS-TLR4-mediated regulation of pAKT, pERK pI-κB, and pMLC20 in LMCs promoted both contractile and inflammatory pathways. Thus, our data provide the first evidence connecting the dynamic changes in innate immune cells on or near the lymphatics and complex cytokine milieu during inflammation with lymphatic dysfunction.

MicroRNA signature of inflamed lymphatic endothelium and role of miR-9 in lymphangiogenesis and inflammation.

The lymphatics have emerged as critical players in the progression and resolution of inflammation. The goal of this study was to identify specific microRNAs (miRNAs) that regulate lymphatic inflammatory processes. Rat mesenteric lymphatic endothelial cells (LECs) were exposed to the proinflammatory cytokine tumor necrosis factor-α for 2, 24, and 96 h, and miRNA profiling was carried out by real-time PCR arrays. Our data demonstrate a specific set of miRNAs that are differentially expressed (>1.8-fold and/or P < 0.05) in LECs in response to tumor necrosis factor-α and are involved in inflammation, angiogenesis, endothelial-mesenchymal transition, and cell proliferation and senescence. We further characterized the expression of miRNA 9 (miR-9) that was induced in LECs and in inflamed rat mesenteric lymphatics. Our results showed that miR-9 overexpression significantly repressed NF-κB expression and, thereby, suppressed inflammation but promoted LEC tube formation, as well as expression of the prolymphangiogenic molecules endothelial nitric oxide synthase and VEGF receptor type 3. LEC viability and proliferation and endothelial-mesenchymal transition were also significantly induced by miR-9. This study provides the first evidence of a distinct profile of miRNAs associated with LECs during inflammation. It also identifies the critical dual role of miR-9 in fine-tuning the balance between lymphatic inflammatory and lymphangiogenic pathways.

Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or ß1-Adrenergic Stimulation.

BACKGROUND: Mammalian hearts exhibit positive inotropic responses to ß-adrenergic stimulation as a consequence of protein kinase A-mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) by protein kinase A accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important because adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure. METHODS AND RESULTS: The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca(2+)]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics because increased pacing or ß1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca(2+)]in transients under each condition were similar to WT. Biochemical measurements confirmed that protein kinase A phosphorylated ser273, ser282, and ser302 in WT cMyBP-C. In contrast, CaMKIIδ, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all. CONCLUSIONS: Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with ß-adrenergic stimulation or increased pacing because of protein kinase A and CaMKIIδ phosphorylations of cMyB-C.

Instability in the central region of tropomyosin modulates the function of its overlapping ends.

The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of contiguous Tm dimers, we used transgenic mice (Tm(DM)) that expressed a mutant α-Tm in the heart; S229E and H276N substitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse cardiac troponin T mutants (TnT(1-44Δ) and TnT(45-74Δ)) that have a divergent effect on the overlapping ends of Tm were employed. The S229E-induced instability in the central region of Tm(DM) altered the overlapping ends of Tm(DM), thereby it negated the attenuating effect of H276N on Ca(2+)-activated maximal tension. The rate of cross-bridge detachment (g) decreased in Tm(DM)+TnT(WT) and Tm(H276N)+TnT(WT) fibers but increased in Tm(DM)+TnT(45-74Δ) fibers; however, TnT(45-74Δ) did not alter g, demonstrating that S229E in Tm(DM) had divergent effects on g. The S229E substitution in Tm(DM) ablated the H276N-induced desensitization of myofilament Ca(2+) sensitivity in Tm(DM)+TnT(1-44Δ) fibers. To our knowledge, novel findings from this study show that the structural instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.

Adenovirus-mediated gene transfection in the isolated lymphatic vessels.

The authors describe technical details of experimental protocol of gene transfection in isolated rat mesenteric lymphatic vessels (MLVs). Authors also refer to the recent publication in Microcirculation, which provides wide set of experimental evidences obtained from confocal microscopic imaging and isolated vessels functional tests, which confirmed a successful achievement of the following goals. (1) Optimization of the experimental conditions to maintain the isolated "normal" rat mesenteric vessels in culture for sufficiently long periods of time to permit effective knockdown or overexpression of selected proteins/genes. (2) Development of the effective transfection protocols for lymphatic muscle and/or endothelial cells in intact isolated rat MLVs without nonspecific impairment on lymphatic contractile function due to the transfection protocol per se.

Structure-function relationships of citrus limonoids on p38 MAP kinase activity in human aortic smooth muscle cells.

Limonoids, abundantly present in citrus fruits, have potential role in reducing risk of different type of cancer. In the present study, we hypothesized that seven structurally different limonoids would involve in inflammatory pathway via modulating p38 MAP kinase activity at various extent in vascular smooth muscle cells. Results demonstrated that the different functional groups containing limonoids had differential effects on the p38 MAP kinase activity in human aortic smooth muscle cells. Among seven limonoids, nomilin exhibited the highest (38%) inhibition of p38 MAP kinase activity, followed by limonin (19%), deacetyl nomilin (19%), and defuran nomilin (17%). While defuran limonin and methyl nomilinate showed no significant decrease in p38 MAP kinase activity, obacunone significantly increased the p38 MAP kinase activity by 38%. Furthermore, TNF-α induced p38 MAP kinase activity in the smooth muscle cells was completely inhibited by nomilin. Thus our data provide the first evidence that nomilin is the potent natural inhibitor for p38 MAP kinase activity in human aortic smooth muscle cells. These data also suggest that a seven-membered A ring with acetoxy group, present in nomilin, seems to be essential for its inhibitory activity on p38 MAP kinase.

Cardiomyocyte contractile status is associated with differences in fibronectin and integrin interactions.

Integrins link the extracellular matrix (ECM) with the intracellular cytoskeleton and other cell adhesion-associated signaling proteins to function as mechanotransducers. However, direct quantitative measurements of the cardiomyocyte mechanical state and its relationship to the interactions between specific ECM proteins and integrins are lacking. The purpose of this study was to characterize the interactions between the ECM protein fibronectin (FN) and integrins in cardiomyocytes and to test the hypothesis that these interactions would vary during contraction and relaxation states in cardiomyocytes. Using atomic force microscopy, we quantified the unbinding force (adhesion force) and adhesion probability between integrins and FN and correlated these measurements with the contractile state as indexed by cell stiffness on freshly isolated mouse cardiomyocytes. Experiments were performed in normal physiological (control), high-K(+) (tonically contracted), or low-Ca(2+) (fully relaxed) solutions. Under control conditions, the initial peak of adhesion force between FN and myocyte alpha(3)beta(1)- and/or alpha(5)beta(1)-integrins was 39.6 +/- 1.3 pN. The binding specificity between FN and alpha(3)beta(1)- and alpha(5)beta(1)-integrins was verified by using monoclonal antibodies against alpha(3)-, alpha(5)-, alpha(3) + alpha(5)-, or beta(1)-integrin subunits, which inhibited binding by 48%, 65%, 70%, or 75%, respectively. Cytochalasin D or 2,3-butanedione monoxime (BDM), to disrupt the actin cytoskeleton or block myofilament function, respectively, significantly decreased the cell stiffness; however, the adhesion force and binding probability were not altered. Tonic contraction with high-K(+) solution increased total cell adhesion (1.2-fold) and cell stiffness (27.5-fold) compared with fully relaxed cells with low-Ca(2+) solution. However, it could be partially prevented by high-K(+) bath solution containing BDM, which suppresses contraction by inhibiting the actin-myosin interactions. Thus, our results demonstrate that integrin binding to FN is modulated by the contractile state of cardiac myocytes.

Methods for lymphatic vessel culture and gene transfection.

OBJECTIVE: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself. METHODS: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3-12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells. RESULTS: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3-12 days without significant impairment of the parameters of their pumping and effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels. CONCLUSIONS: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.

Molecular regulation of lymphatic contractility.

The lymphatic system plays critical roles in body fluid and macromolecular homeostasis, lipid absorption, immune function, and metastasis. To accomplish these tasks, the lymphatics must move lymph and its contents from the interstitial space through the lymph vessels and nodes and into the great veins. Contrary to popular belief, lymph does not passively "drain" down this pathway, because the net pressure gradients oppose flow. Instead, the lymphatics must act as both the conduits that direct and regulate lymph flow and the pumps that generate the lymph flow. Thus, to regulate lymph transport and function, both lymphatic pumping and flow resistance must be controlled. Both of these processes occur via regulation of lymphatic muscle contractions, which are classically thought to occur via the interaction of cell calcium with regulatory and contractile proteins. However, our knowledge of this regulation of lymphatic contractile function is far from complete. In this chapter we review our understanding of the important molecular mechanisms, the calcium regulation, and the contractile/regulatory proteins that control lymphatic contractions. A better understanding of these mechanisms could provide the basis for the development of better diagnostic and treatment modalities for lymphatic dysfunction. While progress has been made in our understanding of the molecular biology of lymphangiogenesis as a result of the development of potential lymphangiogenic therapeutic targets, there are currently no therapeutic agents that specifically modulate lymphatic pump function and lymph flow via lymphatic muscle. However, their development will not be possible until the molecular basis of lymphatic contractility is more fully understood.

Single embryoid body formation in a multi-well plate

Embryoid bodies (EB) formed from murine embryonic stem (ES) cells recapitulate many aspects of a developing embryo. Of specific importance, synchronous differentiation of EB recapitulates organ-specific development and is achieved in culture by formation of uniformly sized EB. The method described here demonstrates a simple and cost-effective way of generating EB from murine ES cells. Single EB are formed in a multi-well plate format and large numbers of EB are generated using a 96-well multi-well plate. Uniform single-sized EB formed in the multi-well are an ideal system for screening compounds and determining differentiation effects. Since EB contain all three germ layers, they are appropriate for studying small molecule effects on differentiation of ES such as is performed in high-throughput screening protocols