Time-restricted feeding attenuates hypercholesterolaemia and atherosclerosis development during circadian disturbance in APOE∗3-Leiden.CETP mice.

BACKGROUND: Circadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has been associated with elevated risk for atherosclerotic cardiovascular disease (asCVD) in humans, but evidence for the effectiveness of prevention strategies is lacking. METHODS: Here, we applied time-restricted feeding (TRF) as a strategy to counteract atherosclerosis development during CD in female APOE∗3-Leiden.CETP mice, a well-established model for humanized lipoprotein metabolism. Control groups were subjected to a fixed 12:12 h light-dark cycle, while CD groups were subjected to 6-h phase advancement every 3 days. Groups had either ad libitum (AL) access to food or were subjected to TRF with restricted food access to the dark phase. FINDINGS: TRF did not prevent the increase in the relative abundance of circulating inflammatory monocytes and elevation of (postprandial) plasma triglycerides during CD. Nonetheless, TRF reduced atherosclerotic lesion size and prevented an elevation in macrophage content of atherosclerotic lesions during CD, while it increased the relative abundance of anti-inflammatory monocytes, prevented activation of T cells, and lowered plasma total cholesterol levels and markers of hepatic cholesterol synthesis. These effects were independent of total food intake. INTERPRETATION: We propose that time restricted eating could be a promising strategy for the primary prevention of asCVD risk in shift workers, which warrants future study in humans. FUNDING: This work was funded by the Novo Nordisk Foundation, the Netherlands Ministry of Social Affairs and Employment, Amsterdam Cardiovascular Sciences, and the Dutch Heart Foundation.

Aging attenuates diurnal lipid uptake by brown adipose tissue.

Brown adipose tissue (BAT) contributes to cardiometabolic health by taking up glucose and lipids for oxidation, a process that displays a strong diurnal rhythm. While aging has been shown to reduce thermogenic characteristics of BAT, it is as yet unknown whether this reduction is specific to the time of day. Therefore, we assessed whole-body and BAT energy metabolism in young and middle-aged male and female C57BL/6J mice and studied the consequences for lipid metabolism in humanized APOE*3-Leiden.CETP mice (also on a C57BL/6J background). We demonstrate that in middle-aged versus young mice body temperature is lower in both male and female mice, while uptake of triglyceride (TG)-derived fatty acids (FAs) by BAT, reflecting metabolic activity, is attenuated at its peak at the onset of the dark (wakeful) phase in female mice. This coincided with delayed plasma clearance of TG-rich lipoproteins and TG-depleted lipoprotein core remnants, and elevated plasma TGs at the same time point. Furthermore, middle-aged female mice showed increased adiposity, accompanied by lipid accumulation, increased expression of genes involved in lipogenesis, and reduced expression of genes involved in fat oxidation and the intracellular clock machinery in BAT. Peak abundance of lipoprotein lipase (LPL), a crucial regulator of FA uptake, was attenuated in BAT. Our findings suggest that LPL is a potential therapeutic target for restoring diurnal metabolic BAT activity, and that efficiency of strategies targeting BAT may be improved by including time of day as an important factor.

von Willebrand Factor: A Central Regulator of Arteriovenous Fistula Maturation Through Smooth Muscle Cell Proliferation and Outward Remodeling.

Background Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal hyperplasia, whereby endothelial cell dysfunction, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation play a crucial role. von Willebrand Factor (vWF) is an endothelial cell-derived protein involved in platelet aggregation and VSMC proliferation. We investigated AVF vascular remodeling in vWF-deficient mice and vWF expression in failed and matured human AVFs. Methods and Results Jugular-carotid AVFs were created in wild-type and vWF-/- mice. AVF flow was determined longitudinally using ultrasonography, whereupon AVFs were harvested 14 days after surgery. VSMCs were isolated from vena cavae to study the effect of vWF on VSMC proliferation. Patient-matched samples of the basilic vein were obtained before brachio-basilic AVF construction and during superficialization or salvage procedure 6 weeks after AVF creation. vWF deficiency reduced VSMC proliferation and macrophage infiltration in the intimal hyperplasia. vWF-/- mice showed reduced outward remodeling (1.5-fold, P=0.002) and intimal hyperplasia (10.2-fold, P<0.0001). AVF flow in wild-type mice was incremental over 2 weeks, whereas flow in vWF-/- mice did not increase, resulting in a two-fold lower flow at 14 days compared with wild-type mice (P=0.016). Outward remodeling in matured patient AVFs coincided with increased local vWF expression in the media of the venous outflow tract. Absence of vWF in the intimal layer correlated with an increase in the intima-media ratio. Conclusions vWF enhances AVF maturation because its positive effect on outward remodeling outweighs its stimulating effect on intimal hyperplasia.

Long-Term Controlled Growth Factor Release Using Layer-by-Layer Assembly for the Development of In Vivo Tissue-Engineered Blood Vessels.

The development of a well-designed tissue-engineered blood vessel (TEBV) still remains a challenge. In recent years, approaches in which the host response to implanted biomaterials is used to generate vascular constructs within the patient's body have gained increasing interest. The delivery of growth factors to these in situ-engineered vascular grafts might enhance myofibroblast recruitment and the secretion of essential extracellular matrix proteins, thereby optimizing their functional properties. Layer-by-layer (LbL) coating has emerged as an innovative technology for the controlled delivery of growth factors in tissue engineering applications. In this study, we combined the use of surface-etched polymeric rods with LbL coatings to control the delivery of TGF-ß1, PDGF-BB, and IGF-1 and steer the foreign body response toward the formation of a functional vascular graft. Results showed that the regenerated tissue is composed of elastin, glycosaminoglycans, and circumferentially oriented collagen fibers, without calcification or systemic spill of the released growth factors. Functional controlled delivery was observed, whereas myofibroblast-rich tissue capsules were formed with enhanced collagen and elastin syntheses using TGF-ß1 and TGF-ß1/PDGF-BB releasing rods, when compared to control rods that were solely surface-engineered by chloroform etching. By combining our optimized LbL method and surface-engineered rods in an in vivo bioreactor approach, we could regulate the fate and ECM composition of in situ-engineered vascular grafts to create a successful in vivo vascular tissue-engineered replacement.

Gene expression changes in the brain of a Cushing's syndrome mouse model.

Excess glucocorticoid exposure affects emotional and cognitive brain functions. The extreme form, Cushing's syndrome, is adequately modelled in the AdKO2.0 mouse, consequential to adrenocortical hypertrophy and hypercorticosteronemia. We previously reported that the AdKO2.0 mouse brain undergoes volumetric changes that resemble closely those of Cushing's syndrome human patients, as well as changes in expression of glial related marker proteins. In the present work, the expression of genes related to glial and neuronal cell populations and functions was assessed in regions of the anterior brain, hippocampus, amygdala and hypothalamus. Glucocorticoid target genes were consistently regulated, including CRH mRNA suppression in the hypothalamus and induction in amygdala and hippocampus, even if glucocorticoid receptor protein was downregulated. Expression of glial genes was also affected in the AdKO2.0 mouse brain, indicating a different activation status in glial cells. Generic markers for neuronal cell populations, and cellular integrity were only slightly affected. Our findings highlight the vulnerability of glial cell populations to chronic high levels of circulating glucocorticoids.

EPH receptor B2 stimulates human monocyte adhesion and migration independently of its EphrinB ligands.

The molecular basis of atherosclerosis is not fully understood and mice studies have shown that Ephrins and EPH receptors play a role in the atherosclerotic process. We set out to assess the role for monocytic EPHB2 and its Ephrin ligands in human atherosclerosis and show a role for EPHB2 in monocyte functions independently of its EphrinB ligands. Immunohistochemical staining of human aortic sections at different stages of atherosclerosis showed that EPHB2 and its ligand EphrinB are expressed in atherosclerotic plaques and that expression proportionally increases with plaque severity. Functionally, stimulation with EPHB2 did not affect endothelial barrier function, nor did stimulation with EphrinB1 or EphrinB2 affect monocyte-endothelial interactions. In contrast, reduced expression of EPHB2 in monocytes resulted in decreased monocyte adhesion to endothelial cells and a decrease in monocyte transmigration, mediated by an altered morphology and a decreased ability to phosphorylate FAK. Our results suggest that EPHB2 expression in monocytes results in monocyte accumulation by virtue of an increase of transendothelial migration, which can subsequently contribute to atherosclerotic plaque progression.

Liposomal Delivery Improves the Efficacy of Prednisolone to Attenuate Renal Inflammation in a Mouse Model of Acute Renal Allograft Rejection.

BACKGROUND: Systemic exposure to high-dose corticosteroids effectively combats acute rejection after kidney transplantation, but at the cost of substantial side effects. In this study, a murine acute renal allograft rejection model was used to investigate whether liposomal-encapsulated prednisolone (LP) facilitates local exposure to enhance its therapeutic effect. METHODS: Male BalbC recipients received renal allografts from male C57BL/6J donors. Recipients were injected daily with 5 mg/kg cyclosporine A and received either 10 mg/kg prednisolone (P), or LP intravenously on day 0, 3, and 6, or no additional treatment. Functional magnetic resonance imaging (fMRI) was performed on day 6 to study allograft perfusion and organs were retrieved on day 7 for further analysis. RESULTS: Staining of polyethylene-glycol-labeled liposomes and high performance liquid chromatography analysis revealed accumulation in the LP treated allograft. LP treatment induced the expression of glucocorticoid responsive gene Fkbp5 in the allograft. Flow-cytometry of allografts revealed liposome presence in CD45 cells, and reduced numbers of F4/80 macrophages, and CD3 T-lymphocytes upon LP treatment. Banff scoring showed reduced interstitial inflammation and tubulitis and fMRI analysis revealed improved allograft perfusion in LP versus NA mice. CONCLUSIONS: Liposomal delivery of prednisolone improved renal bio-availability, increased perfusion and reduced cellular infiltrate in the allograft, when compared with conventional prednisolone. Clinical studies should reveal if treatment with LP results in improved efficacy and reduced side effects in patients with renal allograft rejection.

Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos.

Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the ext2 gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.

Indirect Burst Pressure Measurements for the Mechanical Assessment of Biological Vessels.

IMPACT STATEMENT: Vascular tissue engineering (VTE) is a rapidly expanding field, with numerous approaches being explored both in preclinical and clinical settings. A pivotal factor in the development of VTE techniques is patient safety, notably with respect to the mechanical properties of the vessels. Of the mechanical properties, the bursting strength, representing the ability of a vessel to withstand the forces exerted on it by blood pressure, is the most important. The burst pressure is commonly assessed using one of three methods proposed by the ISO 7198. In this study, we evaluate the three burst pressure assessment methods exactly as they are presently in the field of VTE. We show that the indirect assessment methods, as they are presently used, provide inconsistent and therefore unreliable estimates of the true yield stress of a vessel.

Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.

Arteriovenous access dysfunction is a major cause of morbidity for hemodialysis patients. The pathophysiology of arteriovenous fistula (AVF) maturation failure is associated with inflammation, impaired outward remodeling (OR) and intimal hyperplasia. RP105 is a critical physiologic regulator of TLR4 signaling in numerous cell types. In the present study, we investigated the impact of RP105 on AVF maturation, and defined cell-specific effects of RP105 on macrophages and vascular smooth muscle cells (VSMCs). Overall, RP105-/- mice displayed a 26% decrease in venous OR. The inflammatory response in RP105-/- mice was characterized by accumulation of anti-inflammatory macrophages, a 76% decrease in pro- inflammatory macrophages, a 70% reduction in T-cells and a 50% decrease in MMP-activity. In vitro, anti-inflammatory macrophages from RP105-/- mice displayed increased IL10 production, while MCP1 and IL6 levels secreted by pro-inflammatory macrophages were elevated. VSMC content in RP105-/- AVFs was markedly decreased. In vitro, RP105-/- venous VSMCs proliferation was 50% lower, whereas arterial VSMCs displayed a 50% decrease in migration, relative to WT. In conclusion, the impaired venous OR in RP105-/- mice could result from of a shift in both macrophages and VSMCs towards a regenerative phenotype, identifying a novel relationship between inflammation and VSMC function in AVF maturation.

Development and evaluation of in vivo tissue engineered blood vessels in a porcine model.

BACKGROUND: There's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, we present a novel approach to generate autologous TEBV in vivo. Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature. METHODS AND RESULTS: Rods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting. CONCLUSIONS: Autologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting.

Two-photon intravital multicolour imaging to study metastatic behaviour of cancer cells in vivo.

In the last decade, intravital microscopy on breast tumours in mice at single-cell resolution has resulted in important new insight into mechanisms of metastatic behaviour such as migration, invasion, and intravasation of tumour cells; angiogenesis; and the response of immune cells. This chapter describes the methods that can be used for analysing tumour cell motility in a mouse model of breast cancer metastasis. It includes protocols for generation of a labelled primary tumour, its imaging with two-photon microscopy, and the processing of time-lapse image data. Furthermore, we present a methodology, recently developed in our laboratory that combines multicolour imaging with an inducible cell model to study the role of a specific gene of interest in tumour cell motility in vivo. This protocol can be used to image the metastatic behaviour of different individual tumour cells within the same tumour microenvironment and correlate it with metastasis formation. Additional protocols for labelling macrophages to visualise blood flow and image analysis are also included.

Two-photon intravital multicolor imaging combined with inducible gene expression to distinguish metastatic behavior of breast cancer cells in vivo.

PURPOSE: The aim of this study is to use multicolor intravital imaging together with an inducible cell model to compare metastatic behavior of control and genetically modified breast cancer cell populations within the intact primary tumor of a mouse. PROCEDURE: GFP-MTLn3-ErbB1 cells were generated with doxycycline-regulated conditional transgene expression using lentiviral TREAutoR3-cyan fluorescent protein (CFP). CFP expression together with tumor cell motility is monitored in vitro and in vivo. RESULTS: Effective and tight control of doxycycline-induced CFP expression was observed both in vitro and in vivo. Intravital multiphoton microscopy on intact orthotopic tumors allowed a clear discrimination between GFP-only and (GFP + CFP) cell populations, which enables direct comparison of the motility behavior of two different cell populations in the same microenvironment in vivo. CONCLUSIONS: This system is robust and versatile for conditional gene expression and can be used to study the role of individual candidate metastasis genes in vitro and in vivo. This technology will allow investigations of cellular events in cancer metastasis and in particular intravasation within a primary tumor.

Annexin A1 regulates TGF-beta signaling and promotes metastasis formation of basal-like breast cancer cells.

Annexin A1 (AnxA1) is a candidate regulator of the epithelial- to mesenchymal (EMT)-like phenotypic switch, a pivotal event in breast cancer progression. We show here that AnxA1 expression is associated with a highly invasive basal-like breast cancer subtype both in a panel of human breast cancer cell lines as in breast cancer patients and that AnxA1 is functionally related to breast cancer progression. AnxA1 knockdown in invasive basal-like breast cancer cells reduced the number of spontaneous lung metastasis, whereas additional expression of AnxA1 enhanced metastatic spread. AnxA1 promotes metastasis formation by enhancing TGFbeta/Smad signaling and actin reorganization, which facilitates an EMT-like switch, thereby allowing efficient cell migration and invasion of metastatic breast cancer cells.

Complete focal adhesion kinase deficiency in the mammary gland causes ductal dilation and aberrant branching morphogenesis through defects in Rho kinase-dependent cell contractility.

The adult, virgin mammary gland is a highly organized branched ductal network comprising two major cell types: myoepithelial and luminal epithelial cells. To study the role and mechanism of focal adhesion kinase (FAK)-mediated signaling in mammary gland development and differentiation, we used a conditional Fak-knockout mammary epithelial cell (MEC) transplantation model. Conditional Cre recombinase (Cre)-mediated Fak deletion in primary cultured MECs isolated from FAK(lox/lox)/Rosa26Cre-ERT2 donor mice caused loss of FAK in all mammary cells. Transplantation of Fak-knockout MECs in a cleared mammary fat pad of immune-deficient recipient mice resulted in development of new but dilated virgin ducts with a disrupted myoepithelial and luminal epithelial cell multilayer and aberrant ductal morphogenesis during pregnancy. In the absence of FAK, MECs spread poorly, showed enhanced Rho kinase (ROCK)-mediated cytoskeletal contractility, and failed to respond to receptor-mediated cytoskeletal remodeling. Likewise, FAK deficiency fully inhibited branching morphogenesis of mammary gland organoids in a ROCK-dependent manner. Altogether these data suggest a model in which FAK coordinates contractile forces in MECs to maintain the bilayered cellular organization of myoepithelial and luminal epithelial cells in ducts, thus allowing proper mammary gland development and function.

An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2(-/-) gammac (-/-) mouse.

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2(-/-)gammac(-/-) mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2(-/-) gammac(-/-) mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation.

Integrin signaling via RGD peptides and anti-beta1 antibodies confers resistance to apoptosis in islets of Langerhans.

Islet transplantation is associated with a high rate of early graft failure caused by early immune attack and poor functionality of islets. Apoptosis of islet cells appears soon after islet isolation and primarily involves the beta-cell. The purpose of this study was to determine the effect of ligation to extracellular matrix (ECM) proteins on survival of the islets of Langerhans following islet isolation. Islets that had been cultured for 24 h on collagen type I showed an islet survival of 59.7 +/- 8.7%, while islets that had been cultured on collagen type IV and laminin showed an islet survival of 88.6 +/- 10.3 and 94.3 +/- 5.6%, respectively. Islets that had been pretreated with anti-beta1 antibodies and argenin-glycin-aspartic acid (RGD) peptides showed a decrease in the level of apoptosis by a factor of 2.5 and 3.1, respectively, and an increase of phospho-Akt Ser 473 activity by a factor of 3.1 and 2.9, respectively, compared with untreated islets. When detached from their natural ECM surrounding in the pancreas, islet cells undergo apoptosis, unless islets are cultured on collagen IV or laminin or treated with anti-beta1 integrin antibodies or RGD peptides to mimic ECM ligation. These results indicate that inhibition of anoikis may offer opportunities to improve function and viability of islet cells.

RGD peptides confer survival to hepatocytes via the beta1-integrin-ILK-pAkt pathway.

BACKGROUND/AIMS: Allogeneic cell transplantation is characterized by a lack of sustained survival of the transplanted cells in the recipient. Activation of the appropriate integrin-linked signaling pathways in cells can promote cell survival. The purpose of this study was to determine how presence or absence of anti-beta1 integrin chain antibodies or RGD peptides affects the survival of hepatocytes. METHODS: Hepatocytes of BN rats were isolated. Hepatocyte survival was tested after the hepatocytes had been cultured in the presence of anti-beta1 integrin antibodies or RGD peptides. Hepatocytes that had been given a different treatment were stained for caspase 3 (apoptosis marker) and phospho-Akt Ser 473 (survival marker) and were measured for their integrin-linked kinase (ILK) activity. RESULTS: Ligation of integrins using antibodies against the beta1 integrin chain or RGD peptides protected isolated hepatocytes from apoptosis and resulted in an increased ILK activity and persistent phosphorylation of protein kinase B/Akt at serine 473. CONCLUSIONS: Integrin activation in isolated hepatocytes contributes to the activation of ILK, phosphorylation of Akt on serine residue 473, and inhibition of apoptosis. Integrin signaling through the ILK-phospho Akt pathway protects isolated hepatocytes from apoptosis. This notion may potentially be applied to render the transplantation of hepatocytes more effective.