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.

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.

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.

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.

Integrin αV ß3 can substitute for collagen-binding ß1 -integrins in vivo to maintain a homeostatic interstitial fluid pressure.

NEW FINDINGS: What is the central question of this study? Collagen-binding ß1 -integrins function physiologically in cellular control of dermal interstitial fluid pressure (PIF ) in vivo and thereby participate in control of extravascular fluid volume. During anaphylaxis, simulated by injection of compound 48/80, integrin αV ß3 takes over this physiological function. Here we addressed the question whether integrin αV ß3 can replace collagen-binding ß1 -integrin to maintain a long-term homeostatic PIF . What is the main finding and its importance? Mice lacking the collagen-binding integrin α11 ß1 show a complex dermal phenotype with regard to the interstitial physiology apparent in the control of PIF . Notably dermal PIF is not lowered with compound 48/80 in these animals. Our present data imply that integrin αV ß3 is the likely candidate that has taken over the role of collagen-binding ß1 -integrins for maintaining a steady-state homeostatic PIF . A better understanding of molecular processes involved in control of PIF is instrumental for establishing novel treatment regimens for control of oedema formation in anaphylaxis and septic shock. ABSTRACT: Accumulated data indicate that cell-mediated contraction of reconstituted collagenous gels in vitro can serve as a model for cell-mediated control of interstitial fluid pressure (PIF ) in vivo. A central role for collagen-binding ß1 -integrins in both processes has been established. Furthermore, integrin αV ß3 takes over the role of collagen-binding ß1 -integrins in mediating contraction after perturbations of collagen-binding ß1 -integrins in vitro. Integrin αV ß3 is also instrumental for normalization of dermal PIF that has been lowered due to mast cell degranulation with compound 48/80 (C48/80) in vivo. Here we demonstrate a role of integrin αV ß3 in maintaining a long term homeostatic dermal PIF in mice lacking the collagen-binding integrin  α11 ß1 (α11-/- mice). Measurements of PIF were performed after circulatory arrest. Furthermore, cell-mediated integrin αV ß3 -directed contraction of collagenous gels in vitro depends on free access to a collagen site known to bind several extracellular matrix (ECM) proteins that form substrates for αV ß3 -directed cell attachment, such as fibronectin and fibrin. A streptococcal collagen-binding protein, CNE, specifically binds to and blocks this site on the collagen triple helix. Here we show that whereas CNE perturbed αV ß3 -directed and platelet-derived growth factor BB-induced normalization of dermal PIF after C48/80, it did not affect αV ß3 -dependent maintenance of a homeostatic dermal PIF . These data imply that dynamic modification of the ECM structure is needed during acute patho-physiological modulations of PIF but not for long-term maintenance of a homeostatic PIF . Our data thus show that collagen-binding ß1 -integrins, integrin αV ß3 and ECM structure are potential targets for novel therapy aimed at modulating oedema formation and hypovolemic shock during anaphylaxis.

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.

The notochord in Atlantic salmon (Salmo salar L.) undergoes profound morphological and mechanical changes during development.

We present the development of the notochord of the Atlantic salmon (Salmo salar L.), from early embryo to sexually mature fish. Over the salmon's lifespan, profound morphological changes occur. Cells and gross structures of the notochord reorganize twice. In the embryo, the volume of the notochord is dominated by large, vacuolated chordocytes; each cell can be modeled as a hydrostat organized into a larger cellular-hydrostat network, structurally bound together with desmosomes. After the embryo hatches and grows into a fry, vacuolated chordocytes disappear, replaced by extracellular lacunae. The formation of mineralized, segmental chordacentra stiffens the notochord and creates intervertebral joints, where tissue strain during lateral bending is now focused. As development proceeds towards the parr stage, a process of devacuolization and intracellular filament accumulation occur, forming highly dense, non-vacuolated chordocytes. As extracellular lacunae enlarge, they are enclosed by dense filamentous chordocytes that form transverse intervertebral septa, which are connected to the intervertebral ligaments, and a longitudinal notochordal strand. In the vertebral column of pelagic adults, large vacuolated chordocytes reappear; cells of this secondary population have a volume up to 19 000 times larger than the primary vacuolated chordocytes of the early notochord. In adults the lacunae have diminished in relative size. Hydrostatic pressure within the notochord increases significantly during growth, from 525 Pa in the alevins to 11 500 Pa in adults, at a rate of increase with total body length greater than that expected by static stress similarity. Pressure and morphometric measurements were combined to estimate the stress in the extracellular material of the notochordal sheath and intervertebral ligaments and the flexural stiffness of the axial skeleton. The functional significance of the morphological changes in the axial skeleton is discussed in relation to the different developmental stages and locomotor behavior changes over the lifespan of the fish.

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.

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.

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.

Genetic Engineering of Lymphangiogenesis in Skin Does Not Affect Blood Pressure in Mouse Models of Salt-Sensitive Hypertension.

BACKGROUND: Recent studies have indicated that sodium storage is influenced by macrophages that secrete VEGF-C (vascular endothelial growth factor) during salt stress thus stimulating lymphangiogenesis, thereby acting as a buffer against increased blood pressure (BP). We aimed to explore the role of dermal lymphatics in BP and sodium homeostasis. Our hypothesis was that mice with reduced dermal lymphatic vessels were more prone to develop salt-sensitive hypertension, and that mice with hyperplastic vessels were protected. METHODS: Mice with either hypoplastic (Chy), absent (K14-VEGFR3 [vascular endothelial growth factor receptor 3]-Ig), or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls were given high-salt diet (4% NaCl in the chow), deoxycorticosterone acetate (DOCA)-salt diet and 1% saline to drink or nitric oxide blocker diet L-NG-nitro arginine methyl ester (followed by high salt diet). BP was measured by telemetric recording, and tissue sodium content by ion chromatography. RESULTS: In contrast to previous studies, high salt diet did not induce an increase in BP or sodium storage in any of the mouse strains investigated. DOCA-salt, on the other hand, gave an increase in BP in Chy and K14-VEGFR3-Ig not different from their corresponding WT controls. DOCA induced salt storage in skin and muscle, but to the same extent in mice with dysfunctional lymphatic vessels and WT controls. Lymph flow as assessed by tracer washout was not affected by the diet in any of the mouse strains. CONCLUSIONS: Our results suggest that dermal lymphatic vessels are not involved in salt storage or blood pressure regulation in these mouse models of salt-sensitive hypertension.

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.

Pharmacokinetics and Pharmacodynamics of T-Cell Bispecifics in the Tumour Interstitial Fluid.

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies.

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 x 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.

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.

Improved Drug Delivery to Brain Metastases by Peptide-Mediated Permeabilization of the Blood-Brain Barrier.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood-brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.