Olga Yu. Gasheva

Contraction-initiated NO-dependent lymphatic relaxation: a self-regulatory mechanism in rat thoracic duct

The objectives of this study were to evaluate the physiological importance of the flow and shear generated by phasic contractions of lymphatic vessels and the mechanisms responsible for the influences of such shear on lymphatic pumping. Lymphatic segments of the rat thoracic duct were isolated, cannulated and pressurized. The diastolic diameters were measured in phasically non‐active segments. The diastolic and systolic diameters, half‐relaxation time (HRT), contraction frequency, ejection fraction and fractional pump flow were determined in phasically active segments. Since imposed flow was excluded, flow and shear occurred only as a result of the intrinsic contractions in phasically active segments whereas in phasically non‐active segments contraction‐generated flow and shear were absent. The influences of incrementally increased transmural pressure (from 1 to 5 cmH2O) were examined in control conditions and after NO synthase blockade (l‐NAME 10−4m) or cyclooxygenase blockade (indomethacin 10−5m). The spontaneous phasic contractions produced a flow‐dependent diastolic relaxation. This reduction of the lymphatic tone is a regulatory mechanism that maintains pumping in thoracic duct in an energy‐saving/efficient mode: it improves diastolic filling (enhanced lusitropy – lowering HRT), makes lymphatic contractions stronger (enhanced inotropy – higher contraction amplitude) and propels more fluid forward during each contraction (elevated ejection fraction) while decreasing contraction frequency (reduced chronotropy). The findings also demonstrated that the NO pathway, not the cyclooxygenase pathway is responsible for this reduction of lymphatic tone and is the prevailing pathway responsible for the self‐regulatory adjustment of thoracic duct pumping to changes in lymph flow pattern.

Phasic contractions of rat mesenteric lymphatics increase basal and phasic nitric oxide generation in vivo

Multiple investigators have shown interdependence of lymphatic contractions on nitric oxide (NO) activity by pharmacological and traumatic suppression of endothelial NO synthase (eNOS). We demonstrated that lymphatic diastolic relaxation is particularly sensitive to NO from the lymphatic endothelium. The predicted mechanism is shear forces produced by the lymph flow during phasic pumping, activating eNOS in the lymphatic endothelium to produce NO. We measured [NO] during phasic contractions using microelectrodes on in situ mesenteric lymphatics in anesthetized rats under basal conditions and with an intravenous saline bolus (0.5 ml/100 g) or infusion (0.5 ml x 100 g(-1) x h(-1)). Under basal conditions, [NO] measured on the tubular portions of the lymphatics was approximately 200-250 nM, slightly higher than in the adjacent adipocyte microvasculature, whereas [NO] measured on the lymphatic bulb surface was approximately 400 nM. Immunohistochemistry of eNOS in isolated lympathics indicated a much greater expression in the lymph valves and surrounding bulb area than in the tubular regions. During phasic lymphatic contractions, the valve and tubular [NO] increased with each contraction, and during intravenous saline infusion, [NO] increased in proportion to the contraction frequency and, presumably, lymph flow. The partial blockade of eNOS over approximately 1 cm length with N(omega)-nitro-L-arginine methyl ester lowered the [NO]. These in vivo data document for the first time that both valvular and tubular lymphatic segments increase NO generation during each phasic contraction and that [NO] summated with increased contraction frequency. The combined data predict regional variations in eNOS and [NO] in the tubular and valve areas, plus the summated NO responses dependent on contraction frequency provide for a complex relaxation mechanism involving NO.

Nitric Oxide Formation by Lymphatic Bulb and Valves Is a Major Regulatory Component of Lymphatic Pumping

Microscopic lymphatics produce nitric oxide (NO) during contraction as flow shear activates the endothelial cells. The valve leaflets and bulbous valve housing contain a large amount of endothelial nitric oxide synthase (eNOS) due both to many endothelial cells and increased expression of eNOS. Direct NO measurements indicate the valve area has a 30-50% higher NO concentration ([NO]) than tubular regions although both regions generate equivalent relative increases in [NO] with each contraction. We hypothesize that 1) the greater eNOS and [NO] of the bulb region would have greater effects to lower pumping activity of the overall lymphatic than occurs in tubular regions and 2), the elevated [NO] in the bulb region may be because of high NO production in the valve leaflets that diffuses to the wall of the bulb. Measurement of [NO] with a micropipette inside the lymphatic bulb revealed the valve leaflets generate ~50% larger [NO] than the bulb wall in the in vivo rat mesenteric lymphatics. The valves add NO to the lymph that quickly diffuses to the bulb wall. Bradykinin locally released iontophoretically from a micropipette on both bulbs and tubes increased the [NO] in a dose-dependent manner up to ~50%, demonstrating agonist activation of the NO pathway. However, pumping output determined by contraction frequency and stroke volume decreased much more for the bulb than tubular areas in response to the bradykinin. In effect, NO generation by the bulb area and its valves limits the pumped flow of the total lymphatic by lowering frequency and stroke volume of individual contractions.

Age-Related Alterations of Active Pumping Mechanisms in Rat Thoracic Duct

Objective: To evaluate the age‐related changes in active pumping in thoracic duct (TD) from 24‐month‐old Fisher‐344 rats comparing with TD pumping in 9‐month rats.

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.

Cyclic guanosine monophosphate and the dependent protein kinase regulate lymphatic contractility in rat thoracic duct.

•  Nitric oxide plays a principal role in the lymphatic endothelium/shear‐dependent regulation of contractility in rat thoracic duct. •  In this study we tested the hypothesis that cyclic guanosine monophosphate (cGMP) and the dependent protein kinase (PKG) are central to the intrinsic and extrinsic flow‐dependent modulation of lymphatic contractility. •  Both PKG‐Iα and ‐Iβ isoforms are found in the thoracic duct, with ∼10 times greater expression of the PKG‐Iα protein compared with the aorta and vena cava. •  Functional data demonstrate that cGMP is critical to the flow‐dependent regulation of thoracic duct contractility. •  These findings indicate an important role for PKG, especially PKG‐Iα in these processes and identifies the PKG protein as a potential therapeutic target.

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.

Mast cells and histamine are triggering the NF-κB-mediated reactions of adult and aged perilymphatic mesenteric tissues to acute inflammation

This study aimed to establish mechanistic links between the aging-associated changes in the functional status of mast cells and the altered responses of mesenteric tissue and mesenteric lymphatic vessels (MLVs) to acute inflammation. We used an in vivo model of acute peritoneal inflammation induced by lipopolysaccharide treatment of adult (9-month) and aged (24-month) F-344 rats. We analyzed contractility of isolated MLVs, mast cell activation, activation of nuclear factor-κB (NF-κB) without and with stabilization of mast cells by cromolyn or blockade of all types of histamine receptors and production of 27 major pro-inflammatory cytokines in adult and aged perilymphatic mesenteric tissues and blood. We found that the reactivity of aged contracting lymphatic vessels to LPS-induced acute inflammation was abolished and that activated mast cells trigger NF-κB signaling in the mesentery through release of histamine. The aging-associated basal activation of mesenteric mast cells limits acute inflammatory NF-κB activation in aged mesentery. We conclude that proper functioning of the mast cell/histamine/NF-κB axis is necessary for reactions of the lymphatic vessels to acute inflammatory stimuli as well as for interaction and trafficking of immune cells near and within the collecting lymphatics.

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.

MHC class II invariant chain peptide or gamma-delta T cell depletion ameliorates experimental preeclampsia

Excessive innate immune system activation and inflammation during pregnancy can lead to organ injury and dysfunction and preeclampsia (PE); however, the molecular mechanisms involved are unknown. We tested the hypothesis that Toll-like receptor (TLR) activation induces major histocompatibility complex (MHC) class II invariant chain peptide (CLIP) expression on immune cells, makes them pro-inflammatory, and are necessary to cause PE-like features in mice. Treatment with VG1177, a competitive antagonist peptide for CLIP in the groove of MHC class II, was able to both prevent and treat PE-like features in mice. We then determined that γ-δ T cells are critical for the development of PE-like features in mice since γ-δ T-cell knockout mice, like CLIP deficient mice, are resistant to developing PE-like features. Placentas from women with PE exhibit significantly increased levels of γ-δ T cells. These preclinical data demonstrate that CLIP expression and activated γ-δ T cells are responsible for the development of immunologic PE-like features and that temporarily antagonizing CLIP and/or γ-δ T cells may be a therapeutic strategy for PE.