David C. Zawieja

Inhibition of the active lymph pump by flow in rat mesenteric lymphatics and thoracic duct

There are only a few reports of the influence of imposed flow on an active lymph pump under conditions of controlled intraluminal pressure. Thus, the mechanisms are not clearly defined. Rat mesenteric lymphatics and thoracic ducts were isolated, cannulated and pressurized. Input and output pressures were adjusted to impose various flows. Lymphatic systolic and diastolic diameters were measured and used to determine contraction frequency and pump flow indices. Imposed flow inhibited the active lymph pump in both mesenteric lymphatics and in the thoracic duct. The active pump of the thoracic duct appeared more sensitive to flow than did the active pump of the mesenteric lymphatics. Imposed flow reduced the frequency and amplitude of the contractions and accordingly the active pump flow. Flow‐induced inhibition of the active lymph pump followed two temporal patterns. The first pattern was a rapidly developing inhibition of contraction frequency. Upon imposition of flow, the contraction frequency immediately fell and then partially recovered over time during continued flow. This effect was dependent on the magnitude of imposed flow, but did not depend on the direction of flow. The effect also depended upon the rate of change in the direction of flow. The second pattern was a slowly developing reduction of the amplitude of the lymphatic contractions, which increased over time during continued flow. The inhibition of contraction amplitude was dependent on the direction of the imposed flow, but independent of the magnitude of flow. Nitric oxide was partly but not completely responsible for the influence of flow on the mesenteric lymph pump. Exposure to NO mimicked the effects of flow, and inhibition of the NO synthase by NG‐monomethyl‐l‐arginine attenuated but did not completely abolish the effects of flow.

Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP–dependent proteolysis in 3-dimensional collagen matrices

Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.

Contractile physiology of lymphatics.

The lymphatic system has important roles in body fluid regulation, macromolecular homeostasis, lipid absorption, and immune function. To accomplish these roles, lymphatics must move fluid and its other contents (macromolecules, lipids/chylomicra, immune cells) from the interstitium through the lymphatics, across the nodes, and into the great veins. Thus, the principal task of the lymphatic vascular system is transport. The body must impart energy to the lymph via pumping mechanisms to propel it along the lymphatic network and use pumps and valves to generate lymph flow and prevent its backflow. The lymphatic system utilizes both extrinsic pumps, which rely on the cyclical compression and expansion of lymphatics by surrounding tissue forces, and intrinsic pumps, which rely on the intrinsic rapid/phasic contractions of lymphatic muscle. The intrinsic lymph pump function can be modulated by neural, humoral, and physical factors. Generally, increased lymph pressure/stretch of the muscular lymphatics activates the intrinsic lymph pump, while increased lymph flow/shear in the muscular lymphatics can either activate or inhibit the intrinsic lymph pump depending on the pattern and magnitude of the flow. To regulate lymph transport, lymphatic pumping and resistance must be controlled. A better understanding of these mechanisms could provide the basis for the development of better diagnostic and treatment modalities for lymphatic dysfunction.

Inflammation induces lymphangiogenesis through up-regulation of VEGFR-3 mediated by NF-κB and Prox1

The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-kappaB) family; however, the role of NF-kappaB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-kappaB pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-kappaB followed by sequential up-regulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-kappaB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells, resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-kappaB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-kappaB pathway by inflammatory stimuli activates Prox1, and both NF-kappaB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of preexisting lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability

We previously demonstrated that vascular endothelial growth factor (VEGF)-elicited increase in the permeability of coronary venules was blocked by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). The aim of this study was to delineate in more detail the signaling pathways upstream from NO production in VEGF-induced venular hyperpermeability. The apparent permeability coefficient of albumin (Pa) and endothelial cytosolic Ca2+ concentration ([Ca2+]i) were measured in intact perfused porcine coronary venules using fluorescence microscopy. VEGF (10(-10) M) induced a two- to threefold increase in Pa, which was blocked by a monoclonal antibody directed against the VEGF receptor Flk-1/KDR, the phospholipase C (PLC) antagonist U-73122, or the protein kinase C (PKC) antagonist bisindolylmaleimide (BIM). In 12 venules that displayed the [Ca2+]i response to bradykinin (10(-6) M) and ionomycin (10(-6) M), only 4 vessels responded to VEGF with a transient increase in [Ca2+]i. Furthermore, Western blot analysis of cultured human umbilical vein endothelial cells showed that VEGF increased tyrosine phosphorylation of PLC-gamma and serine phosphorylation of endothelial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC-gamma was greatly attenuated by the KDR receptor antibody and U-73122, but not by BIM or L-NMMA. In contrast, U-73122 and BIM were able to inhibit VEGF-elicited serine phosphorylation of ecNOS. The results suggest that VEGF induces venular hyperpermeability through a KDR receptor-mediated activation of PLC. In turn, ecNOS is activated by PLC-mediated PKC and/or cytosolic Ca2+ elevation stimulation.

Lymph Flow, Shear Stress, and Lymphocyte Velocity in Rat Mesenteric Prenodal Lymphatics

Objective: To measure lymphocyte velocity, lymphatic contraction, and shear stress in phasically contracting lymphatics in situ.

Regional variations of contractile activity in isolated rat lymphatics.

Objective: To evaluate lymphatic contractile activity in different regions of the lymphatic system in a single animal model (the rat thoracic duct, mesenteric, cervical, and femoral lymphatics) in response to changes in lymph pressure and flow.

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.

Molecular and functional analyses of the contractile apparatus in lymphatic muscle

Lymphatics are necessary for the generation and regulation of lymph flow. Lymphatics use phasic contractions and extrinsic compressions to generate flow; tonic contractions alter resistance. Lymphatic muscle exhibits important differences from typical vascular smooth muscle. In this study, the thoracic duct exhibited significant functional differences from mesenteric lymphatics. To understand the molecular basis for these differences, we examined the profiles of contractile proteins and their messages in mesenteric lymphatics, thoracic duct, and arterioles. Results demonstrated that mesenteric lymphatics express only SMB smooth muscle myosin heavy chain (SM‐MHC), whereas thoracic duct and arterioles expressed both SMA and SMB isoforms. Both SM1 and SM2 isoforms of SM‐MHC were detected in arterioles and mesenteric and thoracic lymphatics. In addition, the fetal cardiac/skeletal slow‐twitch muscle‐specific β‐MHC message was detected only in mesenteric lymphatics. All four actin messages, cardiac α‐actin, vascular α‐actin, enteric γ‐actin, and skeletal α‐actin, were present in both mesenteric lymphatics and arterioles. However, in thoracic duct, predominantly cardiac α‐actin and vascular α‐actin were found. Western blot and immunohistochemical analyses corroborated the mRNA studies. However, in arterioles only vascular α‐actin protein was detected. These data indicate that lymphatics display genotypic and phenotypic characteristics of vascular, cardiac, and visceral myocytes, which are needed to fulfill the unique roles of the lymphatic system.

Roles of phosphorylation of myosin binding protein-C and troponin I in mouse cardiac muscle twitch dynamics.

A normal heart increases its contractile force with increasing heart rate. Although calcium handling and myofibrillar proteins have been implicated in maintaining this positive force–frequency relationship (FFR), the exact mechanisms by which it occurs have not been addressed. In this study, we have developed an analytical method to define the calcium–force loop data, which characterizes the function of the contractile proteins in response to calcium that is independent of the calcium handling proteins. Results demonstrate that increasing the stimulation frequency causes increased force production per unit calcium concentration and decreased frequency‐dependent calcium sensitivity during the relaxation phase. We hypothesize that phosphorylation of myosin binding protein‐C (MyBP‐C) and troponin I (TnI) acts coordinately to change the rates of force generation and relaxation, respectively. To test this hypothesis, we performed simultaneous calcium and force measurements on stimulated intact mouse papillary bundles before and after inhibition of MyBP‐C and TnI phosphorylation using the calcium/calmodulin kinase II (CaMK2) inhibitor autocamtide‐2 related inhibitory peptide, or the protein kinase A (PKA) inhibitor 14–22 amide. CaMK2 inhibition reduced both MyBP‐C and TnI phosphorylation and decreased active force without changing the magnitude of the [Ca2+]i transient. This reduced the normalized change in force per change in calcium by 19–39%. Data analyses demonstrated that CaMK2 inhibition changed the myofilament characteristics via a crossbridge feedback mechanism. These results strongly suggest that the phosphorylation of  MyBP‐C and TnI contributes significantly to the rates of force development and relaxation.