Quantification of the Whole Lymph Node Vasculature Based on Tomography of the Vessel Corrosion Casts.

Lymph nodes (LN) are crucial for immune function, and comprise an important interface between the blood and lymphatic systems. Blood vessels (BV) in LN are highly specialized, featuring high endothelial venules across which most of the resident lymphocytes crossed. Previous measurements of overall lymph and BV flow rates demonstrated that fluid also crosses BV walls, and that this is important for immune function. However, the spatial distribution of the BV in LN has not been quantified to the degree necessary to analyse the distribution of transmural fluid movement. In this study, we seek to quantify the spatial localization of LNBV, and to predict fluid movement across BV walls. MicroCT imaging of murine popliteal LN showed that capillaries were responsible for approximately 75% of the BV wall surface area, and that this was mostly distributed around the periphery of the node. We then modelled blood flow through the BV to obtain spatially resolved hydrostatic pressures, which were then combined with Starling's law to predict transmural flow. Much of the total 10 nL/min transmural flow (under normal conditions) was concentrated in the periphery, corresponding closely with surface area distribution. These results provide important insights into the inner workings of LN, and provide a basis for further exploration of the role of LN flow patterns in normal and pathological functions.

Proliferation dynamics of acute myeloid leukaemia and haematopoietic progenitors competing for bone marrow space.

Leukaemia progressively invades bone marrow (BM), outcompeting healthy haematopoiesis by mechanisms that are not fully understood. Combining cell number measurements with a short-timescale dual pulse labelling method, we simultaneously determine the proliferation dynamics of primitive haematopoietic compartments and acute myeloid leukaemia (AML). We observe an unchanging proportion of AML cells entering S phase per hour throughout disease progression, with substantial BM egress at high levels of infiltration. For healthy haematopoiesis, we find haematopoietic stem cells (HSCs) make a significant contribution to cell production, but we phenotypically identify a quiescent subpopulation with enhanced engraftment ability. During AML progression, we observe that multipotent progenitors maintain a constant proportion entering S phase per hour, despite a dramatic decrease in the overall population size. Primitive populations are lost from BM with kinetics that are consistent with ousting irrespective of cell cycle state, with the exception of the quiescent HSC subpopulation, which is more resistant to elimination.

Osteopontin is an autocrine mediator of hepatocyte growth factor-induced invasive growth.

In epithelial cells, hepatocyte growth factor (HGF) activates a genetic program involving cell-cell dissociation ("scattering"), growth and invasiveness. The full program is not elicited by other growth factors like epidermal growth factor, and is aberrantly activated during cancer progression to the invasive-metastatic phenotype. To identify genes involved in the onset of invasive growth, we explored by cDNA microarrays the in vitro transcriptional response to HGF of mouse embryo liver cells. We identified osteopontin (OPN), a secreted matrix protein, as a major HGF transcriptional target. The wave of OPN induction is maximal at 6 h, in concomitance with the initiation of scattering, and is specific, because no other matrix protein among those explored by the microarray is affected. Interestingly, HGF, but not epidermal growth factor, promotes cell adhesion to OPN via the CD44 receptor. Scattering is significantly impaired by antibodies against OPN and CD44; conversely, constitutive OPN overexpression dramatically increases the motile and invasive responses to HGF, leading to disruption of the ordered morphogenetic program triggered by this ligand.