Intracellular calcium dynamics of lymphatic endothelial and muscle cells co-cultured in a Lymphangion-Chip under pulsatile flow.

The lymphatic vascular function is regulated by pulsatile shear stresses through signaling mediated by intracellular calcium [Ca2+]i. Further, the intracellular calcium dynamics mediates signaling between lymphatic endothelial cells (LECs) and muscle cells (LMCs), including the lymphatic tone and contractility. Although calcium signaling has been characterized on LEC monolayers under uniform or step changes in shear stress, these dynamics have not been revealed in LMCs under physiologically-relevant co-culture conditions with LECs or under pulsatile flow. In this study, a cylindrical organ-on-chip platform of the lymphatic vessel (Lymphangion-Chip) consisting of a lumen formed with axially-aligned LECs co-cultured with transversally wrapped layers of LMCs was exposed to step changes or pulsatile shear stress, as often experienced in vivo physiologically or pathologically. Through real-time analysis of intracellular calcium [Ca2+]i release, the device reveals the pulsatile shear-dependent biological coupling between LECs and LMCs. Upon step shear, both cell types undergo a relatively rapid rise in [Ca2+]i followed by a gradual decay. Importantly, under pulsatile flow, analysis of the calcium signal also reveals a secondary sinusoid within the LECs and LMCs that is very close to the flow frequency. Finally, LMCs directly influence the LEC calcium dynamics both under step changes in shear and under pulsatile flow, demonstrating a coupling of LEC-LMC signaling. In conclusion, the Lymphangion-Chip is able to illustrate that intracellular calcium [Ca2+]i in lymphatic vascular cells is dependent on pulsatile shear rate and therefore, serves as an analytical biomarker of mechanotransduction within LECs and LMCs, and functional consequences.

Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 µM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of ß-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure ß-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 µM L-NAME attenuated (p < 0.05) the MPLEC. The ß-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

Lymphatic Cannulation for Lymph Sampling and Molecular Delivery.

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.

Ca2+ release-activated Ca2+ channels are responsible for histamine-induced Ca2+ entry, permeability increase, and interleukin synthesis in lymphatic endothelial cells.

The lymphatic functions in maintaining lymph transport, and immune surveillance can be impaired by infections and inflammation, thereby causing debilitating disorders, such as lymphedema and inflammatory bowel disease. Histamine is a key inflammatory mediator known to trigger vasodilation and vessel hyperpermeability upon binding to its receptors and evoking intracellular Ca2+ ([Ca2+]i) dynamics for downstream signal transductions. However, the exact molecular mechanisms beneath the [Ca2+]i dynamics and the downstream cellular effects have not been elucidated in the lymphatic system. Here, we show that Ca2+ release-activated Ca2+ (CRAC) channels, formed by Orai1 and stromal interaction molecule 1 (STIM1) proteins, are required for the histamine-elicited Ca2+ signaling in human dermal lymphatic endothelial cells (HDLECs). Blockers or antagonists against CRAC channels, phospholipase C, and H1R receptors can all significantly diminish the histamine-evoked [Ca2+]i dynamics in lymphatic endothelial cells (LECs), while short interfering RNA-mediated knockdown of endogenous Orai1 or STIM1 also abolished the Ca2+ entry upon histamine stimulation in LECs. Furthermore, we find that histamine compromises the lymphatic endothelial barrier function by increasing the intercellular permeability and disrupting vascular endothelial-cadherin integrity, which is remarkably attenuated by CRAC channel blockers. Additionally, the upregulated expression of inflammatory cytokines, IL-6 and IL-8, after histamine stimulation was abolished by silencing Orai1 or STIM1 with RNAi in LECs. Taken together, our data demonstrated the essential role of CRAC channels in mediating the [Ca2+]i signaling and downstream endothelial barrier and inflammatory functions induced by histamine in the LECs, suggesting a promising potential to relieve histamine-triggered vascular leakage and inflammatory disorders in the lymphatics by targeting CRAC channel functions.

Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells.

Lymphatic vessels play a critical role in mounting a proper immune response by trafficking peripheral immune cells to draining lymph nodes. Mast cells (MCs) are well known for their roles in type I hypersensitivity reactions, but little is known about their secretory regulation in the lymphatic niche. MCs, as innate sensor and effector cells, reside close to mesenteric lymphatic vessels (MLVs), and their activation and ability to release histamine influences the lymphatic microenvironment in a histamine-NF-κB-dependent manner. Using an established experimental protocol involving surgical isolation of rat mesenteric tissue segments, including MLVs and surrounding perilymphatic tissues, we tested the hypothesis that perilymphatic mesenteric MCs possess histamine receptors (HRs) that bind and respond to the histamine released from these same MCs. Under various experimental conditions, including inflammatory stimulation by LPS, we measured histamine in mesenteric perilymphatic tissues, evaluated expression of histidine decarboxylase in MCs along with the degree of MC degranulation, assessed the functional status of HRs in MCs, and evaluated the ability of histamine itself to induce MC activation. Finally, we evaluated the importance of MCs and HR1 and -2 for MLV-directed trafficking of CD11b/c-positive cells during acute tissue inflammation. Our data indicate the existence of a functionally potent MC-histamine autocrine regulatory loop, the elements of which are crucially important for acute inflammation-induced trafficking of the CD11b/c-positive cells toward MLVs. This MC-histamine loop serves as a first-line cellular servo control system, playing a key role in the innate and adaptive immune response as well as NF-κB-mediated maintenance of body homeostasis.

Prolonged intake of desloratadine: mesenteric lymphatic vessel dysfunction and development of obesity/metabolic syndrome.

This study aimed to establish mechanistic links between the prolonged intake of desloratadine, a common H1 receptor blocker (i.e., antihistamine), and development of obesity and metabolic syndrome. Male Sprague-Dawley rats were treated for 16 wk with desloratadine. We analyzed the dynamics of body weight gain, tissue fat accumulation/density, contractility of isolated mesenteric lymphatic vessels, and levels of blood lipids, glucose, and insulin, together with parameters of liver function. Prolonged intake of desloratadine induced development of an obesity-like phenotype and signs of metabolic syndrome. These alterations in the body included excessive weight gain, increased density of abdominal subcutaneous fat and intracapsular brown fat, high blood triglycerides with an indication of their rerouting toward portal blood, high HDL, high fasting blood glucose with normal fasting and nonfasting insulin levels (insulin resistance), high liver/body weight ratio, and liver steatosis (fatty liver). These changes were associated with dysfunction of mesenteric lymphatic vessels, specifically high lymphatic tone and resistance to flow together with diminished tonic and abolished phasic responses to increases in flow, (i.e., greatly diminished adaptive reserves to respond to postprandial increases in lymph flow). The role of nitric oxide in this flow-dependent adaptation was abolished, with remnants of these responses controlled by lymphatic vessel-derived histamine. Our current data, considered together with reports in the literature, support the notion that millions of the United States population are highly likely affected by underevaluated, lymphatic-related side effects of antihistamines and may develop obesity and metabolic syndrome due to the prolonged intake of this medication. NEW & NOTEWORTHY Prolonged intake of desloratadine induced development of obesity and metabolic syndrome associated with dysfunction of mesenteric lymphatic vessels, high lymphatic tone, and resistance to flow together with greatly diminished adaptive reserves to respond to postprandial increases in lymph flow. Data support the notion that millions of the USA population are highly likely affected by underevaluated, lymphatic-related side effects of antihistamines and may develop obesity and metabolic syndrome due to the prolonged intake of this medication.

Inflammation-induced lymphatic architecture and bone turnover changes are ameliorated by irisin treatment in chronic inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic disease with gastrointestinal dysfunction as well as comorbidities such as inflammation-induced bone loss and impaired immune response. Current treatments for IBD all have negative, potentially severe side effects. We aimed to test whether exogenous treatment with irisin, a novel immunomodulatory adipomyokine, could ameliorate IBD-induced lymphatic and bone alterations. Irisin treatment improved both gut and bone outcomes by mitigating inflammation and restoring structure. In the gut, IBD caused colonic lymphatic hyperproliferation into the mucosal and submucosal compartments. This proliferation in the rodent model is akin to what is observed in IBD patient case studies. In bone, IBD increased osteoclast surface and decreased bone formation. Both gut and osteocytes in bone exhibited elevated levels of TNF-α and receptor activator of NF-κB ligand (RANKL) protein expression. Exogenous irisin treatment restored normal colonic lymphatic architecture and increased bone formation rate concurrent with decreased osteoclast surfaces. After irisin treatment, gut and osteocyte TNF-α and RANKL protein expression levels were no different from vehicle controls. Our data indicate that the systemic immunologic changes that occur in IBD are initiated by damage in the gut and likely linked through the lymphatic system. Additionally, irisin is a potential novel intervention mitigating both local inflammatory changes in the gut and distant changes in bone.-Narayanan, S. A., Metzger, C. E., Bloomfield, S. A., Zawieja, D. C. Inflammation-induced lymphatic architecture and bone turnover changes are ameliorated by irisin treatment in chronic inflammatory bowel disease.

A Novel Computational Model Predicts Key Regulators of Chemokine Gradient Formation in Lymph Nodes and Site-Specific Roles for CCL19 and ACKR4.

The chemokine receptor CCR7 drives leukocyte migration into and within lymph nodes (LNs). It is activated by chemokines CCL19 and CCL21, which are scavenged by the atypical chemokine receptor ACKR4. CCR7-dependent navigation is determined by the distribution of extracellular CCL19 and CCL21, which form concentration gradients at specific microanatomical locations. The mechanisms underpinning the establishment and regulation of these gradients are poorly understood. In this article, we have incorporated multiple biochemical processes describing the CCL19-CCL21-CCR7-ACKR4 network into our model of LN fluid flow to establish a computational model to investigate intranodal chemokine gradients. Importantly, the model recapitulates CCL21 gradients observed experimentally in B cell follicles and interfollicular regions, building confidence in its ability to accurately predict intranodal chemokine distribution. Parameter variation analysis indicates that the directionality of these gradients is robust, but their magnitude is sensitive to these key parameters: chemokine production, diffusivity, matrix binding site availability, and CCR7 abundance. The model indicates that lymph flow shapes intranodal CCL21 gradients, and that CCL19 is functionally important at the boundary between B cell follicles and the T cell area. It also predicts that ACKR4 in LNs prevents CCL19/CCL21 accumulation in efferent lymph, but does not control intranodal gradients. Instead, it attributes the disrupted interfollicular CCL21 gradients observed in Ackr4-deficient LNs to ACKR4 loss upstream. Our novel approach has therefore generated new testable hypotheses and alternative interpretations of experimental data. Moreover, it acts as a framework to investigate gradients at other locations, including those that cannot be visualized experimentally or involve other chemokines.

Collecting lymphatic vessel permeability facilitates adipose tissue inflammation and distribution of antigen to lymph node-homing adipose tissue dendritic cells.

Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. In this study, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived Ags by these cells supported recall T cell responses in the fat and also generated Ag-bearing DCs for emigration into adjacent lymph nodes (LNs). Enhanced recruitment of DCs to inflammation-reactive LNs significantly relied on adipose tissue DCs to maintain sufficient numbers of Ag-bearing DCs as the LN expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for Ag transport into the adjacent LN.

Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome.

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function.

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.

Cinobufotalin impedes Sw.71 cytotrophoblast cell line function via cell cycle arrest and apoptotic signaling.

Preeclampsia (preE) is a hypertensive disorder of pregnancy. Cardiotonic steroids (CTS) are endogenous inhibitors of Na+/K+ ATPase, and at least one CTS, marinobufagenin (MBG), is elevated in a rat model of preE prior to the development of the syndrome. MBG and ouabain impair cytotrophoblast (CTB) cell function, which is critical for placental development. We evaluated the effect of a CTS, cinobufotalin (CINO), on CTB cell function in vitro. CINO at ≥1 nM inhibited CTB cell proliferation, migration, and invasion (p < 0.05), but had no effect on cell viability. There was a higher (p < 0.05) percentage of G0/G1 phase cells in groups treated with CINO at ≥1 nM. CINO caused an increase in stress signaling p38 MAPK and a positive annexin-V staining in CTB cells, indicating the activation of apoptotic signaling. However, the CINO-induced apoptotic signaling was prevented by p38 inhibition. These data demonstrate that CINO impairs CTB cell function via cell cycle arrest and apoptotic signaling.

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.

Stromal interaction molecule 1 (STIM1) and Orai1 mediate histamine-evoked calcium entry and nuclear factor of activated T-cells (NFAT) signaling in human umbilical vein endothelial cells.

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca(2+) mobilization by releasing Ca(2+) from the endoplasmic reticulum and eliciting Ca(2+) entry across the plasma membrane. Herein, we show that histamine-evoked Ca(2+) entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca(2+) release-activated Ca(2+) (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca(2+) entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca(2+) influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca(2+) mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.

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.

Effects of dynamic shear and transmural pressure on wall shear stress sensitivity in collecting lymphatic vessels.

Given the known mechanosensitivity of the lymphatic vasculature, we sought to investigate the effects of dynamic wall shear stress (WSS) on collecting lymphatic vessels while controlling for transmural pressure. Using a previously developed ex vivo lymphatic perfusion system (ELPS) capable of independently controlling both transaxial pressure gradient and average transmural pressure on an isolated lymphatic vessel, we imposed a multitude of flow conditions on rat thoracic ducts, while controlling for transmural pressure and measuring diameter changes. By gradually increasing the imposed flow through a vessel, we determined the WSS at which the vessel first shows sign of contraction inhibition, defining this point as the shear stress sensitivity of the vessel. The shear stress threshold that triggered a contractile response was significantly greater at a transmural pressure of 5 cmH2O (0.97 dyne/cm(2)) than at 3 cmH2O (0.64 dyne/cm(2)). While contraction frequency was reduced when a steady WSS was applied, this inhibition was reversed when the applied WSS oscillated, even though the mean wall shear stresses between the conditions were not significantly different. When the applied oscillatory WSS was large enough, flow itself synchronized the lymphatic contractions to the exact frequency of the applied waveform. Both transmural pressure and the rate of change of WSS have significant impacts on the contractile response of lymphatic vessels to flow. Specifically, time-varying shear stress can alter the inhibition of phasic contraction frequency and even coordinate contractions, providing evidence that dynamic shear could play an important role in the contractile function of collecting lymphatic vessels.

Hyperglycemia down-regulates cGMP-dependent protein kinase I expression in first trimester cytotrophoblast cells.

Diabetes in pregnancy is associated with microvascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be down-regulated under diabetic conditions and contribute to the pathogenesis of vascular complications in diabetes. The present study was undertaken to investigate how high glucose concentrations regulate PKG expression in cytotrophoblast cells (CTBs). Human CTBs (Sw. 71) were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (10 µM SB203580) or 10 µM rosiglitazone. After treatment, the cell lysates were subjected to measure the expression of protein kinase G1α (PKG1α), protein kinase G1ß (PKG1ß), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 ß (sGC1ß) by Western blot. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. The expressions of PKG1α, PKG1ß, sGC1α, and sGC1ß were significantly down-regulated (p < 0.05) in CTBs treated with >135 mg/dL glucose compared to basal (45 mg/dL). The hyperglycemia-induced down-regulation of cGMP and cGMP-dependent PKG were attenuated by the SB203580 or rosiglitazone pretreatment. Exposure of CTBs to excess glucose down-regulates cGMP and cGMP-dependent PKG, contributing to the development of vascular complications in diabetic mothers during pregnancy. The attenuation of hyperglycemia-induced down-regulation of PKG proteins by SB203580 or rosiglitazone pretreatment further suggests the involvement of stress signaling mechanisms in this process.

The effects of inflammatory cytokines on lymphatic endothelial barrier function.

Proper lymphatic function is necessary for the transport of fluids, macromolecules, antigens and immune cells out of the interstitium. The lymphatic endothelium plays important roles in the modulation of lymphatic contractile activity and lymph transport, but it's role as a barrier between the lymph and interstitial compartments is less well understood. Alterations in lymphatic function have long been associated with edema and inflammation although the integrity of the lymphatic endothelial barrier during inflammation is not well-defined. In this paper we evaluated the integrity of the lymphatic barrier in response to inflammatory stimuli commonly associated with increased blood endothelial permeability. We utilized in vitro assays of lymphatic endothelial cell (LEC) monolayer barrier function after treatment with different inflammatory cytokines and signaling molecules including TNF-α, IL-6, IL-1ß, IFN-γ and LPS. Moderate increases in an index of monolayer barrier dysfunction were noted with all treatments (20-60 % increase) except IFN-γ which caused a greater than 2.5-fold increase. Cytokine-induced barrier dysfunction was blocked or reduced by the addition of LNAME, except for IL-1ß and LPS treatments, suggesting a regulatory role for nitric oxide. The decreased LEC barrier was associated with modulation of both intercellular adhesion and intracellular cytoskeletal activation. Cytokine treatments reduced the expression of VE-cadherin and increased scavenging of ß-catenin in the LECs and this was partially reversed by LNAME. Likewise the phosphorylation of myosin light chain 20 at the regulatory serine 19 site, which accompanied the elevated monolayer barrier dysfunction in response to cytokine treatment, was also blunted by LNAME application. This suggests that the lymphatic barrier is regulated during inflammation and that certain inflammatory signals may induce large increases in permeability.

PKC activation increases Ca²âº sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms.

The contractile activity of muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of PKC signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca(2+). It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that the activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca(2+). To test this hypothesis, we determined the effects of PKC activation with phorbol esters [PMA or phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin-permeabilized rat mesenteric and cervical lymphatics or the thoracic duct. The addition of PMA or PDBu induced a significant increase in the contractile force of submaximally activated α-toxin-permeabilized lymphatic muscle independent of a change in intracellular Ca(2+) concentration, and the Ca(2+)-force relationship of lymphatic muscle was significantly left shifted, indicating greater myofilament Ca(2+) sensitivity. Phorbol esters increased the maximal rate of force development, whereas the rate of relaxation was reduced. Western blot and immunohistochemistry data indicated that the initial rapid increase in tension development after stimulation by PDBu was associated with myosin light chain (MLC)20 phosphorylation; however, the later, steady-state Ca(2+) sensitization of permeabilized lymphatic muscle was not associated with increased phosphorylation of MLC20 at Ser(19), 17-kDa C-kinase-potentiated protein phosphatase-1 inhibitor at Thr(38), or caldesmon at Ser(789). Thus, these data indicate that PKC-dependent Ca(2+) sensitization of lymphatic muscle may involve MLC20 phosphorylation-dependent and -independent mechanism(s).

Lymph transport in rat mesenteric lymphatics experiencing edemagenic stress.

OBJECTIVE: To assess lymphatic flow adaptations to edema, we evaluated lymph transport function in rat mesenteric lymphatics under normal and increased fluid volume (edemagenic) conditions in situ. METHODS: Twelve rats were infused with saline (intravenous infusion, 0.2 mL/min/100 g body weight) to induce edema. We intravitally measured mesenteric lymphatic diameter and contraction frequency, as well as lymphocyte velocity and density before, during, and after infusion. RESULTS: A 10-fold increase in lymphocyte velocity (0.1-1 mm/s) and a sixfold increase in flow rate (0.1-0.6 µL/min), were observed post infusion, respectively. There were also increases in contraction frequency and fractional pump flow one minute post infusion. Time-averaged wall shear stress increased 10 fold post infusion to nearly 1.5 dynes/cm(2) . Similarly, maximum shear stress rose from 5 to 40 dynes/cm(2) . CONCLUSIONS: Lymphatic vessels adapted to edemagenic stress by increasing lymph transport. Specifically, the increases in lymphatic contraction frequency, lymphocyte velocity, and shear stress were significant. Lymph pumping increased post infusion, though changes in lymphatic diameter were not statistically significant. These results indicate that edemagenic conditions stimulate lymph transport via increases in lymphatic contraction frequency, lymphocyte velocity, and flow. These changes, consequently, resulted in large increases in wall shear stress, which could then activate NO pathways and modulate lymphatic transport function.