Tine V. Karlsen

A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.

Aspelund et al. discover the presence of a lymphatic vessel network in the dura mater of the mouse brain and show that these dural lymphatic vessels are important for the clearance of macromolecules from the brain.

Immune cells control skin lymphatic electrolyte homeostasis and blood pressure

The skin interstitium sequesters excess Na+ and Cl- in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl- accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl-, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.

Atrial natriuretic peptide modulation of albumin clearance and contrast agent permeability in mouse skeletal muscle and skin: role in regulation of plasma volume

Atrial natriuretic peptide (ANP) via its guanylyl cyclase‐A (GC‐A) receptor participates in regulation of arterial blood pressure and vascular volume. Previous studies demonstrated that concerted renal diuretic/natriuretic and endothelial permeability effects of ANP cooperate in intravascular volume regulation. We show that the microvascular endothelial contribution to the hypovolaemic action of ANP can be measured by the magnitude of the ANP‐induced increase in blood‐to‐tissue albumin transport, measured as plasma albumin clearance corrected for intravascular volume change, relative to the corresponding increase in ANP‐induced renal water excretion. We used a two‐tracer method with isotopically labelled albumin to measure clearances in skin and skeletal muscle of: (i) C57BL6 mice; (ii) mice with endothelium‐restricted deletion of GC‐A (floxed GC‐A × tie2‐Cre: endothelial cell (EC) GC‐A knockout (KO)); and (iii) control littermates (floxed GC‐A mice with normal GC‐A expression levels). Comparison of albumin clearances in hypervolaemic EC GC‐A KO mice with normovolaemic littermates demonstrated that skeletal muscle albumin clearance with ANP treatment accounts for at most 30% of whole body clearance required for ANP to regulate plasma volume. Skin microcirculation responded to ANP similarly. Measurements of permeability to a high molecular mass contrast agent (35 kD Gadomer) by dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) enabled repeated measures in individual animals and confirmed small increases in muscle and skin microvascular permeability after ANP. These quantitative methods will enable further evaluation of the contribution of ANP‐dependent microvascular beds (such as gastro‐intestinal tract) to plasma volume regulation.

Transcapillary fluid balance consequences of missing initial lymphatics studied in a mouse model of primary lymphoedema

To investigate the phenotypic consequences of a deranged lymphangiogenesis in relation to tissue fluid accumulation and the possible role of inflammation in the pathogenesis of lymphoedema, we measured determinants of transcapillary fluid filtration and inflammatory mediators in the interstitial fluid in genetically engineered Chy mice, a model for primary congenital lymphoedema (Milroys disease). Although initial lymphatics were not present in dermis in any of the areas studied (fore paw, hind paw, thigh and back skin) interstitial fluid pressure (Pif), measured with micropipettes, and tissue fluid volumes were significantly increased only in the areas with visible swelling – the fore and hind paw, whereas interstitial colloid osmotic pressure (COPif) was increased in all the skin areas examined. A volume load of 15% of body weight resulted in a more pronounced increase in Pif as well as a four‐fold increase in interstitial fluid volume in Chy relative to wild‐type (wt) mice, showing the quantitative importance of lymphatics for fluid homeostasis during acute perturbations. A similar level of proinflammatory markers in interstitial fluid in early established lymphoedema (3–4 months) in Chy and wt suggests that inflammation does not have a major pathogenetic role for the development of lymphoedema, whereas a reduced level of the immunomodulatory cytokine interleukin (IL)‐4 may result in a reduced immunological defence ability and thus lead to the increase in inflammatory cytokines IL‐2 and IL‐6 observed at a later stage (11–13 months). Our data suggest that primary lymphoedema results in a high interstitial fluid protein concentration that does not induce an interstitial inflammatory reaction per se, and furthermore shows the paramount importance of the initial lymphatics in tissue fluid homeostasis, especially during perturbations of transcapillary fluid balance.

Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin

There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.

A novel function of insulin in rat dermis

In this study we present a novel function of insulin in rat dermis. We investigated local effects of insulin on interstitial fluid pressure (Pif), and capillary albumin leakage and pro‐inflammatory cytokine production in skin and serum after intravenous lipopolysaccharide (LPS), tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) challenge treated with a glucose–insulin–potassium regimen (GIK). The main objective for this study was to investigate anti‐inflammatory effects of insulin. Work by others shows that insulin stimulates cell adhesion, and that this effect is dependent upon phosphatidylinositol 3‐kinase (PI3K) activity. Cytokines like platelet‐derived growth factor BB (PDGF‐BB) attenuate lowering of Pif, possibly via PI3K. LPS and pro‐inflammatory cytokines contribute to oedema development during acute inflammation by lowering the Pif. Intravenous injection of LPS, TNF‐α or IL‐1β to Wistar Møller rats caused a lowering of Pif, but after local injection of insulin in the paw, Pif increased back to control values. IL‐1β caused a lowering in control from −0.5 ± 0.2 mmHg to −3.0 ± 0.2 mmHg after 20 min (mean ±s.e.m.) (P < 0.05). Within 50 min after insulin injection the pressure was increased to −0.6 ± 0.2 mmHg (P > 0.05 compared with control). Insulin was given together with a PI3K inhibitor (wortmannin) locally in the skin, almost abolishing the effect of insulin on Pif. A GIK regimen was given as a continuous intravenous infusion, significantly attenuating the oedema formation after LPS or TNF‐α/IL‐1β challenge. The same GIK regimen caused a significant reduction in pro‐inflammatory cytokines in serum and in interstitial fluid in skin of endotoxaemic rats. These experiments show a possible role for insulin in the interstitium during inflammation induced by LPS and TNF‐α/IL‐1β. Insulin can attenuate a lowering of Pif possibly via PI3K, and it has an anti‐inflammatory effect by inhibiting production of pro‐inflammatory cytokines.

Increased Interstitial Protein Because of Impaired Lymph Drainage Does Not Induce Fibrosis and Inflammation in Lymphedema

Objective—The pathophysiology of lymphedema is incompletely understood. We asked how transcapillary fluid balance parameters and lymph flow are affected in a transgenic mouse model of primary lymphedema, which due to an inhibition of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling lacks dermal lymphatics, and whether protein accumulation in the interstitium occurring in lymphedema results in inflammation. Methods and Results—As estimated using a new optical-imaging technique, we found that this signaling defect resulted in lymph drainage in hind limb skin of K14-VEGFR-3-Ig mice that was 34% of the corresponding value in wild-type. The interstitial fluid pressure and tissue fluid volumes were significantly increased in the areas of visible swelling only, whereas the colloid osmotic pressure in plasma, and thus the colloid osmotic pressure gradient, was reduced compared to wild-type mice. An acute volume load resulted in an exaggerated interstitial fluid pressure response in transgenic mice. There was no accumulation of collagen or lipid in skin, suggesting that chronic edema presented in the K14-VEGFR-3-Ig mouse was not sufficient to induce changes in tissue composition. Proinflammatory cytokines (interleukin-2, interleukin-6, interleukin-12) in subcutaneous interstitial fluid and macrophage infiltration in skin of the paw were lower, whereas the monocyte/macrophage cell fraction in blood and spleen was higher in transgenic compared with wild-type mice. Conclusion—Our data suggest that a high interstitial protein concentration and longstanding edema is not sufficient to induce fibrosis and inflammation characteristic for the human condition and may have implications for our understanding of the pathophysiology of this condition.

Proteomic evaluation of inflammatory proteins in rat spleen interstitial fluid and lymph during LPS-induced systemic inflammation reveals increased levels of ADAMST1.

The spleen is a part of the immune system and is involved in the response to a systemic inflammation induced by blood borne pathogens that may induce sepsis. Knowledge about the protein composition of the spleen microenvironment in a control situation and during systemic inflammation may contribute to our understanding of the pathophysiology of sepsis. To our knowledge, the proteome of the fluid phase of the spleen microenvironment has not previously been investigated. In order to access the proximal fluid surrounding the splenic cells, we collected postnodal efferent spleen lymph from rats by cannulation, and spleen interstitial fluid (IF) by centrifugation. The origin of the isolated spleen IF was assessed by the extracellular tracer (51)Cr-EDTA and the plasma tracer (125)I-HSA. Spleen lymph, IF, and plasma samples were collected during lipopolysaccharide (LPS) induced systemic inflammation and analyzed using a cytokine multiplex assay and, for the first time, using label-free mass spectrometry based proteomics. The concentrations of TNF-α, IL-1β, IL-6, and IL-10 increased severalfold in all fluids after LPS exposure. In total, 281, 201, and 236 proteins were identified in lymph, IF, and plasma, respectively, and several of these were detected after LPS only. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was detected by proteomics (the pro- region) in lymph only after LPS. ADAMTS1 was assessed by ELISA (the metalloproteinase domain), and the concentration was significantly higher in IF and lymph than in plasma in a control situation, showing local production in the spleen. A dramatic increase in ADAMTS1 was detected in lymph, IF, and plasma after LPS exposure. In conclusion, the procedures we used to isolate IF and lymph from the spleen during LPS enabled detection of locally produced proteins. Furthermore, we have demonstrated that the inflammatory proteome is different in the spleen microenvironment when compared to that in plasma.

High-salt diet increases hormonal sensitivity in skin pre-capillary resistance vessels.

Recent data indicate that the skin of rats on a high‐salt diet is able to accumulate Na+ without commensurate water. This extrarenal mechanism of Na+ homoeostasis could affect skin vasoregulation. We hypothesized that the major resistance vessel of rat skin, the pre‐capillary arterioles, has increased vasoreactivity within the physiological range of circulating ANG II, a hormone relevant to salt‐sensitive hypertension.

Fibroblast EXT1-Levels Influence Tumor Cell Proliferation and Migration in Composite Spheroids

Background Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model. Methodology/Principal Findings We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate. Conclusions/Significance This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression.