VEGF-B hypertrophy predisposes to transition from diastolic to systolic heart failure in hypertensive rats.

AIMS: Cardiac energy metabolism is centrally involved in heart failure (HF), although the direction of the metabolic alterations is complex and likely dependent on the particular stage of HF progression. Vascular endothelial growth factor B (VEGF-B) has been shown to modulate metabolic processes and to induce physiological cardiac hypertrophy; thus, it could be cardioprotective in the failing myocardium. This study investigates the role of VEGF-B in cardiac proteomic and metabolic adaptation in HF during aldosterone and high-salt hypertensive challenges. METHODS AND RESULTS: Male rats overexpressing the cardiac-specific VEGF-B transgene (VEGF-B TG) were treated for 3 or 6 weeks with deoxycorticosterone-acetate combined with a high-salt (HS) diet (DOCA + HS) to induce hypertension and cardiac damage. Extensive longitudinal echocardiographic studies of HF progression were conducted, starting at baseline. Sham-treated rats served as controls. To evaluate the metabolic alterations associated with HF, cardiac proteomics by mass spectrometry was performed. Hypertrophic non-treated VEGF-B TG hearts demonstrated high oxygen and adenosine triphosphate (ATP) demand with early onset of diastolic dysfunction. Administration of DOCA + HS to VEGF-B TG rats for 6 weeks amplified the progression from cardiac hypertrophy to HF, with a drastic drop in heart ATP concentration. Dobutamine stress echocardiographic analyses uncovered a significantly impaired systolic reserve. Mechanistically, the hallmark of the failing TG heart was an abnormal energy metabolism with decreased mitochondrial ATP, preceding the attenuated cardiac performance and leading to systolic HF. CONCLUSIONS: This study shows that the VEGF-B TG accelerates metabolic maladaptation which precedes structural cardiomyopathy in experimental hypertension and ultimately leads to systolic HF.

Na+ is shifted from the extracellular to the intracellular compartment and is not inactivated by glycosaminoglycans during high salt conditions in rats.

Recently, studies have emerged suggesting that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. We investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. Na+ accumulation was induced in rats by a high salt diet (HSD) (8% NaCl and 1% saline to drink) or by implantation of a deoxycorticosterone acetate (DOCA) tablet (1% saline to drink) using rats on a low salt diet (LSD) (0.1% NaCl) on tap water as control. Na+ and K+ were assessed by ion chromatography in tissue eluates, and the extracellular volume by equilibration of 51 Cr-EDTA. By tangential sectioning of the skin, we found a low Na+ content and extracellular volume in epidermis, both parameters rising by ∼30% and 100%, respectively, in LSD and even more in HSD and DOCA when entering dermis. We found evidence for an extracellular Na+ gradient from epidermis to dermis shown by an estimated concentration in epidermis ∼2 and 4-5 times that of dermis in HSD and DOCA-salt. There was intracellular storage of Na+ in skin, muscle, and myocardium without a concomitant increase in hydration. Our data suggest that there is a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. Salt stress results in intracellular storage of Na+ in exchange with K+ in skeletal muscle and myocardium that may have electromechanical consequences. KEY POINTS: Studies have suggested that Na+ can be retained or removed without commensurate water retention or loss, and that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. In the present study, we investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. We used two common models for salt-sensitive hypertension: high salt and a deoxycorticosterone salt diet. We found a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. There was intracellular Na+ storage in muscle and myocardium without a concomitant increase in hydration, comprising storage that may have electromechanical consequences in salt stress.

Skin Sodium Accumulates in Psoriasis and Reflects Disease Severity.

Sodium can accumulate in the skin at concentrations exceeding serum levels. A high sodium environment can lead to pathogenic T helper 17 cell expansion. Psoriasis is a chronic inflammatory skin disease in which IL-17‒producing T helper 17 cells play a crucial role. In an observational study, we measured skin sodium content in patients with psoriasis and in age-matched healthy controls by Sodium-23 magnetic resonance imaging. Patients with PASI > 5 showed significantly higher sodium and water content in the skin but not in other tissues than those with lower PASI or healthy controls. Skin sodium concentrations measured by Sodium-23 spectroscopy or by atomic absorption spectrometry in ashed-skin biopsies verified the findings with Sodium-23 magnetic resonance imaging. In vitro T helper 17 cell differentiation of naive CD4+ cells from patients with psoriasis markedly induced IL-17A expression under increased sodium chloride concentrations. The imiquimod-induced psoriasis mouse model replicated the human findings. Extracellular tracer Chromium-51-EDTA measurements in imiquimod- and sham-treated skin showed similar extracellular volumes, rendering excessive water of intracellular origin. Chronic genetic IL-17A‒driven psoriasis mouse models underlined the role of IL-17A in dermal sodium accumulation and inflammation. Our data describe skin sodium as a pathophysiological feature of psoriasis, which could open new avenues for its treatment.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

AIM: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. METHODS: In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. RESULTS: Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. CONCLUSION: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Time course of decompensation after angiotensin II and high-salt diet in Balb/CJ mice suggests pulmonary hypertension-induced cardiorenal syndrome.

The genetic background of a mouse strain determines its susceptibility to disease. C57BL/6J and Balb/CJ are two widely used inbred mouse strains that we found react dramatically differently to angiotensin II and high-salt diet (ANG II + Salt). Balb/CJ show increased mortality associated with anuria and edema formation while C57BL/6J develop arterial hypertension but do not decompensate and die. Clinical symptoms of heart failure in Balb/CJ mice gave the hypothesis that ANG II + Salt impairs cardiac function and induces cardiac remodeling in male Balb/CJ but not in male C57BL/6J mice. To test this hypothesis, we measured cardiac function using echocardiography before treatment and every day for 7 days during treatment with ANG II + Salt. Interestingly, pulsed wave Doppler of pulmonary artery flow indicated increased pulmonary vascular resistance and right ventricle systolic pressure in Balb/CJ mice, already 24 h after ANG II + Salt treatment was started. In addition, Balb/CJ mice showed abnormal diastolic filling indicated by reduced early and late filling and increased isovolumic relaxation time. Furthermore, Balb/CJ exhibited lower cardiac output compared with C57BL/6J even though they retained more sodium and water, as assessed using metabolic cages. Left posterior wall thickness increased during ANG II + Salt treatment but did not differ between the strains. In conclusion, ANG II + Salt treatment causes early restriction of pulmonary flow and reduced left ventricular filling and cardiac output in Balb/CJ, which results in fluid retention and peripheral edema. This makes Balb/CJ a potential model to study the adaptive capacity of the heart for identifying new disease mechanisms and drug targets.

Stromal integrin α11-deficiency reduces interstitial fluid pressure and perturbs collagen structure in triple-negative breast xenograft tumors.

BACKGROUND: Cancer progression is influenced by a pro-tumorigenic microenvironment. The aberrant tumor stroma with increased collagen deposition, contractile fibroblasts and dysfunctional vessels has a major impact on the interstitial fluid pressure (PIF) in most solid tumors. An increased tumor PIF is a barrier to the transport of interstitial fluid into and within the tumor. Therefore, understanding the mechanisms that regulate pressure homeostasis can lead to new insight into breast tumor progression, invasion and response to therapy. The collagen binding integrin α11ß1 is upregulated during myofibroblast differentiation and expressed on fibroblasts in the tumor stroma. As a collagen organizer and a probable link between contractile fibroblasts and the complex collagen network in tumors, integrin α11ß1 could be a potential regulator of tumor PIF. METHODS: We investigated the effect of stromal integrin α11-deficiency on pressure homeostasis, collagen organization and tumor growth using orthotopic and ectopic triple-negative breast cancer xenografts (MDA-MB-231 and MDA-MB-468) in wild type and integrin α11-deficient mice. PIF was measured by the wick-in-needle technique, collagen by Picrosirius Red staining and electron microscopy, and uptake of radioactively labeled 5FU by microdialysis. Further, PIF in heterospheroids composed of MDA-MB-231 cells and wild type or integrin α11-deficient fibroblasts was measured by micropuncture. RESULTS: Stromal integrin α11-deficiency decreased PIF in both the orthotopic breast cancer models. A concomitant perturbed collagen structure was seen, with fewer aligned and thinner fibrils. Integrin α11-deficiency also impeded MDA-MB-231 breast tumor growth, but no effect was observed on drug uptake. No effects were seen in the ectopic model. By investigating the isolated effect of integrin α11-positive fibroblasts on MDA-MB-231 cells in vitro, we provide evidence that PIF regulation was mediated by integrin α11-positive fibroblasts. CONCLUSION: We hereby show the importance of integrin α11ß1 in pressure homeostasis in triple-negative breast tumors, indicating a new role for integrin α11ß1 in the tumor microenvironment. Our data suggest that integrin α11ß1 has a pro-tumorigenic effect on triple-negative breast cancer growth in vivo. The significance of the local microenvironment is shown by the different effects of integrin α11ß1 in the orthotopic and ectopic models, underlining the importance of choosing an appropriate preclinical model.

Angiotensin II and salt-induced decompensation in Balb/CJ mice is aggravated by fluid retention related to low oxidative stress.

Balb/CJ mice are more sensitive to treatment with angiotensin II (ANG II) and high-salt diet compared with C57BL/6J mice. Together with higher mortality, they develop edema, signs of heart failure, and acute kidney injury. The aim of the present study was to identify differences in renal gene regulation that may affect kidney function and fluid balance, which could contribute to decompensation in Balb/CJ mice after ANG II + salt treatment. Male Balb/CJ and C57BL/6J mice were divided into the following five different treatment groups: control, ANG II, salt, ANG II + salt, and ANG II + salt + N-acetylcysteine. Gene expression microarrays were used to explore differential gene expression after treatment and between the strains. Published data from the Mouse Genome Database were used to identify the associated genomic differences. The glomerular filtration rate (GFR) was measured using inulin clearance, and fluid balance was measured using metabolic cages. Gene ontology enrichment analysis of gene expression microarrays identified glutathione transferase (antioxidant system) as highly enriched among differentially expressed genes. Balb/CJ mice had similar GFR compared with C57BL/6J mice but excreted less Na+ and water, although net fluid and electrolyte balance did not differ, suggesting that Balb/CJ mice may be inherently more prone to decompensation. Interestingly, C57BL/6J mice had higher urinary oxidative stress despite their relative protection from decompensation. In addition, treatment with the antioxidant N-acetylcysteine decreased oxidative stress in C57BL/6J mice, reduced urine excretion, and increased mortality. Balb/CJ mice are more sensitive than C57BL/6J to ANG II + salt, in part mediated by lower oxidative stress, which favors fluid and Na+ retention.

High-Salt Diet Causes Expansion of the Lymphatic Network and Increased Lymph Flow in Skin and Muscle of Rats.

Objective- A commonly accepted pivotal mechanism in fluid volume and blood pressure regulation is the parallel relationship between body Na+ and extracellular fluid content. Several recent studies have, however, shown that a considerable amount of Na+ can be retained in skin without commensurate water retention. Here, we asked whether a salt accumulation shown to result in VEGF (vascular endothelial growth factor)-C secretion and lymphangiogenesis had any influence on lymphatic function. Approach and Results- By optical imaging of macromolecular tracer washout in skin, we found that salt accumulation resulted in an increase in lymph flow of 26% that was noticeable only after including an overnight recording period. Surprisingly, lymph flow in skeletal muscle recorded with a new positron emission tomography/computed tomography method was also increased after salt exposure. The transcapillary filtration was unaffected by the high-salt diet and deoxycorticosterone-salt treatment, suggesting that the capillary barrier was not influenced by the salt accumulation. A significant reduction in lymph flow after depletion of macrophages/monocytes by clodronate suggests these cells are involved in the observed lymph flow response, together with collecting vessels shown here to enhance their contraction frequency as a response to extracellular Na+. Conclusions- The observed changes in lymph flow suggest that the lymphatics may influence long-term regulation of tissue fluid balance during salt accumulation by contributing to fluid homeostasis in skin and muscle. Our studies identify lymph clearance as a potential disease-modifying factor that might be targeted in conditions characterized by salt accumulation like chronic kidney disease and salt-sensitive hypertension.

Lymphangiogenesis Facilitates Initial Lymph Formation and Enhances the Dendritic Cell Mobilizing Chemokine CCL21 Without Affecting Migration.

OBJECTIVE: Lymphatic vessels play an important role in body fluid, as well as immune system homeostasis. Although the role of malfunctioning or missing lymphatics has been studied extensively, less is known on the functional consequences of a chronically expanded lymphatic network or lymphangiogenesis. APPROACH AND RESULTS: To this end, we used K14-VEGF-C (keratin-14 vascular endothelial growth factor-C) transgenic mice overexpressing the vascular endothelial growth factor C in skin and investigated the responses to inflammatory and fluid volume challenges. We also recorded interstitial fluid pressure, a major determinant of lymph flow. Transgenic mice had a strongly enhanced lymph vessel area in skin. Acute inflammation induced by lipopolysaccharide and chronic inflammation by delayed-type hypersensitivity both resulted in increased interstitial fluid pressure and reduced lymph flow, both to the same extent in wild-type and transgenic mice. Hyperplastic lymphatic vessels, however, demonstrated enhanced transport capacity after local fluid overload not induced by inflammation. In this situation, interstitial fluid pressure was increased to a similar extent in the 2 strains, thus, suggesting that the enhanced lymph vessel area facilitated initial lymph formation. The increased lymph vessel area resulted in an enhanced production of the chemoattractant CCL21 that, however, did not result in augmented dendritic cell migration after induction of local skin inflammation by fluorescein isothiocyanate. CONCLUSIONS: An expanded lymphatic network is capable of enhanced chemoattractant production, and lymphangiogenesis will facilitate initial lymph formation favoring increased clearance of fluid in situations of augmented fluid filtration.

Interstitial IgG antibody pharmacokinetics assessed by combined in vivo- and physiologically-based pharmacokinetic modelling approaches.

KEY POINTS: For therapeutic antibodies, total tissue concentrations are frequently reported as a lump sum measure of the antibody in residual plasma, interstitial fluid and cells. In terms of correlating antibody exposure to a therapeutic effect, however, interstitial pharmacokinetics might be more relevant. In the present study, we collected total tissue and interstitial antibody biodistribution data in mice and assessed the composition of tissue samples aiming to correct total tissue measurements for plasma and cellular content. All data and parameters were integrated into a refined physiologically-based pharmacokinetic model for monoclonal antibodies to enable the tissue-specific description of antibody pharmacokinetics in the interstitial space. We found that antibody interstitial concentrations are highly tissue-specific and dependent on the underlying capillary structure but, in several tissues, they reach relatively high interstitial concentrations, contradicting the still-prevailing view that both the distribution to tissues and the interstitial concentrations for antibodies are generally low. ABSTRACT: For most therapeutic antibodies, the interstitium is the target space. Although experimental methods for measuring antibody pharmacokinetics (PK) in this space are not well established, thus making quantitative assessment difficult, the interstitial antibody concentration is assumed to be low. In the present study, we combined direct quantification of antibodies in the interstitial fluid with a physiologically-based PK (PBPK) modelling approach, with the aim of better describing the PK of monoclonal antibodies in the interstitial space of different tissues. We isolated interstitial fluid by tissue centrifugation and conducted an antibody biodistribution study in mice, measuring total tissue and interstitial concentrations in selected tissues. Residual plasma, interstitial volumes and lymph flows, which are important PBPK model parameters, were assessed in vivo. We could thereby refine the PBPK modelling of monoclonal antibodies, better interpret antibody biodistribution data and more accurately predict their PK in the different tissue spaces. Our results indicate that, in tissues with discontinuous capillaries (liver and spleen), interstitial concentrations are reflected by the plasma concentration. In tissues with continuous capillaries (e.g. skin and muscle), ∼50-60% of the plasma concentration is found in the interstitial space. In the brain and kidney, on the other hand, antibodies are restricted to the vascular space. Our data may significantly impact the interpretation of biodistribution data of monoclonal antibodies and might be important when relating measured concentrations to a therapeutic effect. By contrast to the view that the antibody distribution to the interstitial space is limited, using direct measurements and model-based data interpretation, we show that high antibody interstitial concentrations are reached in most tissues.

High-Salt Diet Causes Osmotic Gradients and Hyperosmolality in Skin Without Affecting Interstitial Fluid and Lymph.

The common notion is that the body Na+ is maintained within narrow limits for fluid and blood pressure homeostasis. Several studies have, however, shown that considerable amounts of Na+ can be retained or removed from the body without commensurate water loss and that the skin can serve as a major salt reservoir. Our own data from rats have suggested that the skin is hypertonic compared with plasma on salt storage and that this also applies to skin interstitial fluid. Even small electrolyte gradients between plasma and interstitial fluid would represent strong edema-generating forces. Because the water accumulation has been shown to be modest, we decided to reexamine with alternative methods in rats whether interstitial fluid is hypertonic during salt accumulation induced by high-salt diet (8% NaCl and 1% saline to drink) or deoxycorticosterone pellet implantation. These treatments resulted both in increased systemic blood pressure, skin salt, and water accumulation and in skin hyperosmolality. Interstitial fluid isolated from implanted wicks and lymph draining the skin was, however, isosmotic, and Na+ concentration in fluid isolated by centrifugation and in lymph was not different from plasma. Interestingly, by eluting layers of the skin, we could show that there was an osmolality and urea gradient from epidermis to dermis. Collectively, our data suggest that fluid leaving the skin as lymph is isosmotic to plasma but also that the skin can differentially control its own electrolyte microenvironment by creating local gradients that may be functionally important.

Age-related changes in rat dermal extracellular matrix composition affect the distribution of plasma proteins as a function of size and charge.

Collagen and glycosaminoglycans (GAGs) constituting the ECM may limit the space available and thus exclude macromolecules from a fraction of the interstitial fluid (IF) phase. This exclusion phenomenon is of importance for transcapillary fluid and solute exchange. The purpose of the study was to examine the range of interstitial exclusion in rat skin by using probes within a span of molecular weights and electrical charge and also to test if a change in interstitial composition, occurring as a consequence of aging, affected exclusion. To this end, we used a novel approach, involving the exact determination of albumin concentration and mass in IF and tissue eluate by HPLC and thereafter, expressing the corresponding numbers relative to albumin for a set of probe proteins assessed by quantitative proteomics. Albumin was excluded from 55±4% (n=8) of the extracellular fluid phase. There was a highly significant, positive correlation between probe Stokes-Einstein (SE) radius and fractional excluded volume (VEF), described by VEF=0.078·SE radius+0.269 (P<0.001), and oppositely, a negative correlation between probe isoelectric point (pI) and exclusion for proteins with comparable size, VEF=-0.036·pI+0.719 (P=0.04). Aging resulted in a significant reduction in skin hydration and sulfated GAGs, a moderate increase in hyaluronan, and a corresponding, reduced VEF for albumin and the other macromolecular probes. Our findings suggest that the changes in the ECM in aged skin may result in delayed adjustments of fluid perturbations and reduced ability for salt storage.

Fibroblast α11ß1 integrin regulates tensional homeostasis in fibroblast/A549 carcinoma heterospheroids.

We have previously shown that fibroblast expression of α11ß1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11+/+) or with a deletion (α11-/-) in integrin α11 gene. Our data show that α11-/-/A549 spheroids are larger than α11+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11ß1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11ß1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11ß1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.

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.

Isolation of interstitial fluid and demonstration of local proinflammatory cytokine production and increased absorptive gradient in chronic peritoneal dialysis.

In peritoneal dialysis (PD) patients, the frequent exposure to "unphysiological" dialysis fluids elicits a chronic state of a low-grade peritoneal inflammation leading to interstitial matrix remodeling and angiogenesis. Proinflammatory cytokines are important regulators involved in this inflammatory process that ultimately leads to dysfunction of the peritoneum as a dialysis membrane. We aimed to measure the local concentrations of proinflammatory cytokines in the peritoneal interstitial fluid (IF). Furthermore, we wanted to assess how the driving forces for fluid and solute exchanges are affected in a remodeled interstitial matrix and thus measured the colloid osmotic pressure (COP) gradient in rats that were exposed to chronic PD. After 8 wk of peritoneal dialysis, IF from peritoneum was isolated using a centrifugation method, and was analyzed for cytokine content and COP along with plasma. For several of the proinflammatory cytokines there were gradients from IF to plasma, showing local production. For some cytokines, the concentration in IF was increased severalfold, whereas IL-18 was increased systemically due to PD. Furthermore, the presence of the catheter per se seemed to increase cytokine levels. COP in IF was significantly decreased in the PD group, while collagen and hyaluronan content was increased. Collectively, our data suggest that the increased levels of proinflammatory cytokines after PD may be an integral component of the development of fibrosis and angiogenesis commonly seen in PD patients, and the decreased COP in IF after chronic PD may shift the Starling equilibrium across peritoneal capillaries to an absorptive state.

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-1ß, 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.

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.

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.

Lowering of interstitial fluid pressure after neurogenic inflammation in mouse skin is partly dependent on mast cells.

Neurogenic inflammation is known to induce lowering of interstitial fluid pressure (P(if)) in mouse skin. This study examined the possible role of mast cell activation secondary to neuropeptide release in lowering of P(if) by using Kit(W)/Kit(W-v) mice, which are devoid of mast cells, including connective tissue mast cells (CTMCs). P(if) was measured in paw skin of anesthetized (fentanyl-fluanison and midazolam, 1:1) mice with glass capillaries connected to a servo-controlled counterpressure system. In contrast to wild-type mice, intravenous administration of mast cell-activating compound 48/80 induced no lowering of P(if) in Kit(W)/Kit(W-v) mice. Intravenous challenge with substance P (SP), calcitonin gene-related peptide (CGRP), or capsaicin induced a significant (P < 0.05) lowering of P(if) in wild-type mice to -2.16 +/- 0.28, -1.96 +/- 0.11, and -2.22 +/- 0.19 mmHg, respectively, compared with vehicle (-0.49 +/- 0.11 mmHg). In Kit(W)/Kit(W-v) mice the P(if) response to SP was completely abolished (-0.53 +/- 0.32 mmHg) while the response to CGRP and capsaicin was attenuated (-1.33 +/- 0.13 and -1.42 +/- 0.13 mmHg, respectively) although significantly (P < 0.05) lowered compared with vehicle. Immunohistochemical analysis revealed no difference in distribution or density of SP- and CGRP-immunoreactive fibers in paws of Kit(W)/Kit(W-v) compared with wild-type mice. We conclude that lowering of P(if) normally depends on mast cells. However, the sensory nerves can also elicit a lowering of P(if) that is independent of mast cells.

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 (Milroy's 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 (P(if)), 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 (COP(if)) was increased in all the skin areas examined. A volume load of 15% of body weight resulted in a more pronounced increase in P(if) 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.