Isolation of lymph shows dysregulation of STAT3 and CREB pathways in the spleen and liver during leukemia development in a rat model.

BACKGROUND AND AIMS: Acute myeloid leukemia (AML) is a heterogeneous malignant condition characterized by massive infiltration of poorly differentiated white blood cells in the blood stream, bone marrow, and extramedullary sites. During leukemic development, hepatosplenomegaly is expected to occur because large blood volumes are continuously filtered through these organs. We asked whether infiltration of leukemic blasts initiated a response that could be detected in the interstitial fluid phase of the spleen and liver. MATERIAL AND METHODS: We used a rat model known to mimic human AML in growth characteristics and behavior. By cannulating efferent lymphatic vessels from the spleen and liver, we were able to monitor the response of the microenvironment during AML development. RESULTS AND DISCUSSION: Flow cytometric analysis of lymphocytes showed increased STAT3 and CREB signaling in spleen and depressed signaling in liver, and proteins related to these pathways were identified with a different profile in lymph and plasma in AML compared with control. Additionally, several proteins were differently regulated in the microenvironment of spleen and liver in AML when compared with control. CONCLUSION: Interstitial fluid, and its surrogate efferent lymph, can be used to provide unique information about responses in AML-infiltered organs and substances released to the general circulation during leukemia development.

The complexity of kidney disease and diagnosing it - cystatin C, selective glomerular hypofiltration syndromes and proteome regulation.

Estimation of kidney function is often part of daily clinical practice, mostly done by using the endogenous glomerular filtration rate (GFR)-markers creatinine or cystatin C. A recommendation to use both markers in parallel in 2010 has resulted in new knowledge concerning the pathophysiology of kidney disorders by the identification of a new set of kidney disorders, selective glomerular hypofiltration syndromes. These syndromes, connected to strong increases in mortality and morbidity, are characterized by a selective reduction in the glomerular filtration of 5-30 kDa molecules, such as cystatin C, compared to the filtration of small molecules <1 kDa dominating the glomerular filtrate, for example water, urea and creatinine. At least two types of such disorders, shrunken or elongated pore syndrome, are possible according to the pore model for glomerular filtration. Selective glomerular hypofiltration syndromes are prevalent in investigated populations, and patients with these syndromes often display normal measured GFR or creatinine-based GFR-estimates. The syndromes are characterized by proteomic changes promoting the development of atherosclerosis, indicating antibodies and specific receptor-blocking substances as possible new treatment modalities. Presently, the KDIGO guidelines for diagnosing kidney disorders do not recommend cystatin C as a general marker of kidney function and will therefore not allow the identification of a considerable number of patients with selective glomerular hypofiltration syndromes. Furthermore, as cystatin C is uninfluenced by muscle mass, diet or variations in tubular secretion and cystatin C-based GFR-estimation equations do not require controversial race or sex terms, it is obvious that cystatin C should be a part of future KDIGO guidelines.

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.

Abnormal neonatal sodium handling in skin precedes hypertension in the SAME rat.

We discovered high Na+ and water content in the skin of newborn Sprague-Dawley rats, which reduced ~ 2.5-fold by 7 days of age, indicating rapid changes in extracellular volume (ECV). Equivalent changes in ECV post birth were also observed in C57Bl/6 J mice, with a fourfold reduction over 7 days, to approximately adult levels. This established the generality of increased ECV at birth. We investigated early sodium and water handling in neonates from a second rat strain, Fischer, and an Hsd11b2-knockout rat modelling the syndrome of apparent mineralocorticoid excess (SAME). Despite Hsd11b2-/- animals exhibiting lower skin Na+ and water levels than controls at birth, they retained ~ 30% higher Na+ content in their pelts at the expense of K+ thereafter. Hsd11b2-/- neonates exhibited incipient hypokalaemia from 15 days of age and became increasingly polydipsic and polyuric from weaning. As with adults, they excreted a high proportion of ingested Na+ through the kidney, (56.15 ± 8.21% versus control 34.15 ± 8.23%; n = 4; P < 0.0001), suggesting that changes in nephron electrolyte transporters identified in adults, by RNA-seq analysis, occur by 4 weeks of age. Our data reveal that Na+ imbalance in the Hsd11b2-/- neonate leads to excess Na+ storage in skin and incipient hypokalaemia, which, together with increased, glucocorticoid-induced Na+ uptake in the kidney, then contribute to progressive, volume contracted, salt-sensitive hypertension. Skin Na+ plays an important role in the development of SAME but, equally, may play a key physiological role at birth, supporting post-natal growth, as an innate barrier to infection or as a rudimentary kidney.

Interstitial fluid drainage from rat apical area takes place via vessels in the mandibular canal.

We sought to investigate the transport route for protein-rich fluid from the apical area towards the draining lymph nodes. The first mandibular molar root canals in 24 female Wistar rats were instrumented and filled with radioactive-labelled human serum albumin. The rats were sacrificed at different intervals beginning after 10 min (time 0) and continuing up to 72 h. Three jaw segments, gingiva around the first molar, blood samples, submandibular and cervical lymph nodes were collected and analyzed for radioactivity. The starting volume of tracer (control) for all experiments was calculated from measurements at time 0. At time 0, radioactivity was only detected in the jaw segments. Within lymph nodes and serum, the tracer was found after 4 h, with the highest amount recorded in serum up to 24 h. Lymphatics were found within the mandibular canal along blood vessels and nerves and exiting via foramen mandibularis, after immunohistochemical staining in four untreated rats. Our results show tracer distribution from the apical area towards the mandibular canal in a posterior direction. The tracer washout rate was low, and the fluid was mainly absorbed into blood vessels. The lymphatics in the mandibular canal may be more important for immune cell transport than for fluid drainage.

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.

GRHL2 Is Required for Collecting Duct Epithelial Barrier Function and Renal Osmoregulation.

Collecting ducts make up the distal-most tubular segments of the kidney, extending from the cortex, where they connect to the nephron proper, into the medulla, where they release urine into the renal pelvis. During water deprivation, body water preservation is ensured by the selective transepithelial reabsorption of water into the hypertonic medullary interstitium mediated by collecting ducts. The collecting duct epithelium forms tight junctions composed of barrier-enforcing claudins and exhibits a higher transepithelial resistance than other segments of the renal tubule exhibit. However, the functional relevance of this strong collecting duct epithelial barrier is unresolved. Here, we report that collecting duct-specific deletion of an epithelial transcription factor, grainyhead-like 2 (GRHL2), in mice led to reduced expression of tight junction-associated barrier components, reduced collecting duct transepithelial resistance, and defective renal medullary accumulation of sodium and other osmolytes. In vitro, Grhl2-deficient collecting duct cells displayed increased paracellular flux of sodium, chloride, and urea. Consistent with these effects, Grhl2-deficient mice had diabetes insipidus, produced dilute urine, and failed to adequately concentrate their urine after water restriction, resulting in susceptibility to prerenal azotemia. These data indicate a direct functional link between collecting duct epithelial barrier characteristics, which appear to prevent leakage of interstitial osmolytes into urine, and body water homeostasis.

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.

Unilateral renal ischaemia in rats induces a rapid secretion of inflammatory markers to renal lymph and increased capillary permeability.

A better understanding of the inflammatory process associated with renal ischaemia-reperfusion (IR) injury may be clinically important. In this study we examined the role of the kidney in production of inflammatory mediators by analysing renal lymph after 30 min unilateral occlusion of renal artery followed by 120 min reperfusion, as well as the effect of IR on size selectivity for proteins in both glomerular and peritubular capillaries. All measured mediators increased dramatically in renal hilar lymph, plasma and renal cortical tissue samples and returned to control levels after 120 min reperfusion. The responses were differentiated; interleukin-1ß, monocyte chemoattractant protein-1 and leptin were markedly increased in plasma before reperfusion, reflecting an extrarenal response possibly induced by afferent renal nerve activity from the ischaemic kidney. Tumour necrosis factor-α was the only mediator showing elevated lymph-to-plasma ratio following 30 min reperfusion, indicating that most cytokines were released directly into the bloodstream. The IR-induced rise in cytokine levels was paralleled by a significant increase in high molecular weight plasma proteins in both lymph and urine. The latter was shown as a 14- to 166-fold increase in glomerular sieving coefficient of plasma proteins assessed by a novel proteomic approach, and indicated a temporarily reduced size selectivity of both glomerular and peritubular capillaries. Collectively, our data suggest that cytokines from the ischaemic kidney explain most of the rise in plasma concentration, and that the locally produced substances enter the systemic circulation through transport directly to plasma and not via the interstitium to lymph.

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.

Distribution volumes of macromolecules in human ovarian and endometrial cancers--effects of extracellular matrix structure.

Elements of the extracellular matrix (ECM), notably collagen and glucosaminoglycans, will restrict part of the space available for soluble macromolecules simply because the molecules cannot occupy the same space. This phenomenon may influence macromolecular drug uptake. To study the influence of steric and charge effects of the ECM on the distribution volumes of macromolecules in human healthy and malignant gynecologic tissues we used as probes 15 abundant plasma proteins quantified by high-resolution mass spectrometry. The available distribution volume (VA) of albumin was increased in ovarian carcinoma compared with healthy ovarian tissue. Furthermore, VA of plasma proteins between 40 and 190 kDa decreased with size for endometrial carcinoma and healthy ovarian tissue, but was independent of molecular weight for the ovarian carcinomas. An effect of charge on distribution volume was only found in healthy ovaries, which had lower hydration and high collagen content, indicating that a condensed interstitium increases the influence of negative charges. A number of earlier suggested biomarker candidates were detected in increased amounts in malignant tissue, e.g., stathmin and spindlin-1, showing that interstitial fluid, even when unfractionated, can be a valuable source for tissue-specific proteins. We demonstrate that the distribution of abundant plasma proteins in the interstitium can be elucidated by mass spectrometry methods and depends markedly on hydration and ECM structure. Our data can be used in modeling of drug uptake, and give indications on ECM components to be targeted to increase the uptake of macromolecular substances.

Interstitial fluid-a reflection of the tumor cell microenvironment and secretome.

The interstitium or interstitial space describes the space outside the blood and lymphatic vessels. It contains two phases; the interstitial fluid (IF) and the extracellular matrix. In this review we focus on the interstitial fluid phase, which is the physical and biochemical microenvironment of the cells, and more specifically that of tumors. IF is created by transcapillary filtration and cleared by lymphatic vessels, and contains substances that are either produced and secreted locally, thus denoted secretome, or brought to the organ by the circulation. The structure of the interstitium is discussed briefly and moreover techniques for IF isolation focusing on those that are relevant for studies of the secretome. Accumulated data show that tumor IF is hypoxic and acidic compared with subcutaneous IF and plasma, and that there are gradients between IF and plasma giving information on where substances are produced and thereby reflecting the local microenvironment. We review recent data on the origin of tissue specific substances, challenges related to isolating a representative secretome and the use of this as a substrate for biomarker identification. Finally we perform a comparative analysis across human tumor types and techniques and show that there is great variation in the results obtained that may at least partially be due to the isolation method used. We conclude that when care is taken in isolation of substrate, analysis of the secretome may give valuable biological insight and result in identification of biomarker candidates. This article is part of a Special Issue entitled: An Updated Secretome.

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue.

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.

Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats.

BACKGROUND: It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. METHODS: In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography-tandem mass spectrometry. RESULTS: 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. CONCLUSIONS: Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats.

Effects of hydration on steric and electric charge-induced interstitial volume exclusion--a model.

The presence of collagen and charged macromolecules like glycosaminoglycans (GAGs) in the interstitial space limits the space available for plasma proteins and other macromolecules. This phenomenon, known as interstitial exclusion, is of importance for interstitial fluid volume regulation. Physical/mathematical models are presented for calculating the exclusion of electrically charged and neutral macromolecules that equilibrate in the interstitium under various degrees of hydration. Here, a central hypothesis is that the swelling of highly electrically charged GAGs with increased hydration shields parts of the neutral collagen of the interstitial matrix from interacting with electrically charged macromolecules, such that exclusion of charged macromolecules exhibits change due to steric and charge effects. GAGs are also thought to allow relatively small neutral, but also charged macromolecules neutralized by a very high ionic strength, diffuse into the interior of GAGs, whereas larger macromolecules may not. Thus, in the model, relatively small electrically charged macromolecules, such as human serum albumin, and larger neutral macromolecules such as IgG, will have quite similar total volume exclusion properties in the interstitium. Our results are in agreement with ex vivo and in vivo experiments, and suggest that the charge of GAGs or macromolecular drugs may be targeted to increase the tissue uptake of macromolecular therapeutic agents.

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.

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.

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.

Access to the spleen microenvironment through lymph shows local cytokine production, increased cell flux, and altered signaling of immune cells during lipopolysaccharide-induced acute inflammation.

The spleen is involved in fluid volume regulation, immune responses, and hematopoiesis. Yet, the composition of the fluid phase within the spleen microenviroment, the migratory routes of lymphocytes as well as the splenic response to bacterial endotoxin is incomplete. To address these issues, we isolated postnodal lymph in rats by cannulating an efferent lymphatic draining the spleen, and assessed the secretion of signaling substances during a septic response induced by LPS. Spleen lymph flow increased 8-fold after LPS exposure. The spleen exhibited a permeable microvasculature with low sieving of macromolecules that was absent after exposure to LPS. Furthermore, after LPS exposure the spleen contributed significantly to the production of pro- and anti-inflammatory cytokines, and experiments in splenectomized rats suggested it may induce a protracted inflammation because of a dominant role in IL-6 production. A significant amount of lymphocytes exited via lymphatics draining the spleen in control rats. LPS-induced inflammation resulted in increased T cell and reduced B cell subset fractions, and gave a significant increase in CD4(+) and CD8(+) subset T cell efflux and a reduced B cell efflux in spleen lymph. Exposure of leukocytes to the spleen microenvironment affected their signaling status, and by phosphorylation specific flow cytometry we could identify STAT3 and CREB as important mediators in the cellular signaling occurring during endotoxemia. We conclude that analysis of spleen lymph may unravel immune cell migration patterns and local signaling, and immune cells exit via lymph having acquired specific activation signatures after exposure to the spleen microenvironment.