An inducible Cldn11-CreERT2 mouse line for selective targeting of lymphatic valves.

Luminal valves of collecting lymphatic vessels are critical for maintaining unidirectional flow of lymph and their dysfunction underlies several forms of primary lymphedema. Here, we report on the generation of a transgenic mouse expressing the tamoxifen inducible CreERT2 under the control of Cldn11 promoter that allows, for the first time, selective and temporally controlled targeting of lymphatic valve endothelial cells. We show that within the vasculature CLDN11 is specifically expressed in lymphatic valves but is not required for their development as mice with a global loss of Cldn11 display normal valves in the mesentery. Tamoxifen treated Cldn11-CreERT2 mice also carrying a fluorescent Cre-reporter displayed reporter protein expression selectively in lymphatic valves and, to a lower degree, in venous valves. Analysis of developing vasculature further showed that Cldn11-CreERT2 -mediated recombination is induced during valve leaflet formation, and efficient labeling of valve endothelial cells was observed in mature valves. The Cldn11-CreERT2 mouse thus provides a valuable tool for functional studies of valves.

Combined transcriptome and proteome profiling of the pancreatic ß-cell response to palmitate unveils key pathways of ß-cell lipotoxicity.

BACKGROUND: Prolonged exposure to elevated free fatty acids induces ß-cell failure (lipotoxicity) and contributes to the pathogenesis of type 2 diabetes. In vitro exposure of ß-cells to the saturated free fatty acid palmitate is a valuable model of lipotoxicity, reproducing features of ß-cell failure observed in type 2 diabetes. In order to map the ß-cell response to lipotoxicity, we combined RNA-sequencing of palmitate-treated human islets with iTRAQ proteomics of insulin-secreting INS-1E cells following a time course exposure to palmitate. RESULTS: Crossing transcriptome and proteome of palmitate-treated ß-cells revealed 85 upregulated and 122 downregulated genes at both transcript and protein level. Pathway analysis identified lipid metabolism, oxidative stress, amino-acid metabolism and cell cycle pathways among the most enriched palmitate-modified pathways. Palmitate induced gene expression changes compatible with increased free fatty acid mitochondrial import and ß-oxidation, decreased lipogenesis and modified cholesterol transport. Palmitate modified genes regulating endoplasmic reticulum (ER) function, ER-to-Golgi transport and ER stress pathways. Furthermore, palmitate modulated cAMP/protein kinase A (PKA) signaling, inhibiting expression of PKA anchoring proteins and downregulating the GLP-1 receptor. SLC7 family amino-acid transporters were upregulated in response to palmitate but this induction did not contribute to ß-cell demise. To unravel critical mediators of lipotoxicity upstream of the palmitate-modified genes, we identified overrepresented transcription factor binding sites and performed network inference analysis. These identified LXR, PPARα, FOXO1 and BACH1 as key transcription factors orchestrating the metabolic and oxidative stress responses to palmitate. CONCLUSIONS: This is the first study to combine transcriptomic and sensitive time course proteomic profiling of palmitate-exposed ß-cells. Our results provide comprehensive insight into gene and protein expression changes, corroborating and expanding beyond previous findings. The identification of critical drivers and pathways of the ß-cell lipotoxic response points to novel therapeutic targets for type 2 diabetes.

Smooth muscle cell recruitment to lymphatic vessels requires PDGFB and impacts vessel size but not identity.

Tissue fluid drains through blind-ended lymphatic capillaries, via smooth muscle cell (SMC)-covered collecting vessels into venous circulation. Both defective SMC recruitment to collecting vessels and ectopic recruitment to lymphatic capillaries are thought to contribute to vessel failure, leading to lymphedema. However, mechanisms controlling lymphatic SMC recruitment and its role in vessel maturation are unknown. Here, we demonstrate that platelet-derived growth factor B (PDGFB) regulates lymphatic SMC recruitment in multiple vascular beds. PDGFB is selectively expressed by lymphatic endothelial cells (LECs) of collecting vessels. LEC-specific deletion of Pdgfb prevented SMC recruitment causing dilation and failure of pulsatile contraction of collecting vessels. However, vessel remodelling and identity were unaffected. Unexpectedly, Pdgfb overexpression in LECs did not induce SMC recruitment to capillaries. This was explained by the demonstrated requirement of PDGFB extracellular matrix (ECM) retention for lymphatic SMC recruitment, and the low presence of PDGFB-binding ECM components around lymphatic capillaries. These results demonstrate the requirement of LEC-autonomous PDGFB expression and retention for SMC recruitment to lymphatic vessels, and suggest an ECM-controlled checkpoint that prevents SMC investment of capillaries, which is a common feature in lymphedematous skin.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes.

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved ß-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Cathelicidins positively regulate pancreatic ß-cell functions.

Cathelicidins are pleiotropic antimicrobial peptides largely described for innate antimicrobial defenses and, more recently, immunomodulation. They are shown to modulate a variety of immune or nonimmune host cell responses. However, how cathelicidins are expressed by ß cells and modulate ß-cell functions under steady-state or proinflammatory conditions are unknown. We find that cathelicidin-related antimicrobial peptide (CRAMP) is constitutively expressed by rat insulinoma ß-cell clone INS-1 832/13. CRAMP expression is inducible by butyrate or phenylbutyric acid and its secretion triggered upon inflammatory challenges by IL-1ß or LPS. CRAMP promotes ß-cell survival in vitro via the epidermal growth factor receptor (EGFR) and by modulating expression of antiapoptotic Bcl-2 family proteins: p-Bad, Bcl-2, and Bcl-xL. Also via EGFR, CRAMP stimulates glucose-stimulated insulin secretion ex vivo by rat islets. A similar effect is observed in diabetes-prone nonobese diabetic (NOD) mice. Additional investigation under inflammatory conditions reveals that CRAMP modulates inflammatory responses and ß-cell apoptosis, as measured by prostaglandin E2 production, cyclooxygenases (COXs), and caspase activation. Finally, CRAMP-deficient cnlp(-/-) mice exhibit defective insulin secretion, and administration of CRAMP to prediabetic NOD mice improves blood glucose clearance upon glucose challenge. Our finding suggests that cathelicidins positively regulate ß-cell functions and may be potentially used for intervening ß-cell dysfunction-associated diseases.

Glucocorticoids suppress GLP-1 secretion: possible contribution to their diabetogenic effects.

Evidence indicates that subtle abnormalities in GC (glucocorticoid) plasma concentrations and/or in tissue sensitivity to GCs are important in the metabolic syndrome, and it is generally agreed that GCs induce insulin resistance. In addition, it was recently reported that short-term exposure to GCs reduced the insulinotropic effects of the incretin GLP-1 (glucagon-like peptide 1). However, although defective GLP-1 secretion has been correlated with insulin resistance, potential direct effects of GCs on GLP-1-producing L-cell function in terms of GLP-1 secretion and apoptosis have not been studied in any greater detail. In the present study, we sought to determine whether GCs could exert direct effects on GLP-1-producing L-cells in terms of GLP-1 secretion and cell viability. We demonstrate that the GR (glucocorticoid receptor) is expressed in GLP-1-producing cells, where GR activation in response to dexamethasone induces SGK1 (serum- and glucocorticoid-inducible kinase 1) expression, but did not influence preproglucagon expression or cell viability. In addition, dexamethasone treatment of enteroendocrine GLUTag cells reduced GLP-1 secretion induced by glucose, 2-deoxy-D-glucose, fructose and potassium, whereas the secretory response to a phorbol ester was unaltered. Furthermore, in vivo administration of dexamethasone to rats reduced the circulating levels of GLP-1 concurrent with induction of insulin resistance and glucose intolerance. We can conclude that GR activation in GLP-1-producing cells will diminish the secretory responsiveness of these cells to subsequent carbohydrate stimulation. These effects may not only elucidate the pathogenesis of steroid diabetes, but could ultimately contribute to the identification of novel molecular targets for controlling incretin secretion.

Inhibition of palmitate-induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6).

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta-cells, an event linked to apoptotic loss of ß-cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage-inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin-producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA-activated protein kinase-like ER-localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate-induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin-producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels.

Penile cavernous sinusoids are Prox1-positive hybrid vessels.

Endothelial cells (ECs) of blood and lymphatic vessels have distinct identity markers that define their specialized functions. Recently, hybrid vasculatures with both blood and lymphatic vessel-specific features have been discovered in multiple tissues. Here, we identify the penile cavernous sinusoidal vessels (pc-Ss) as a new hybrid vascular bed expressing key lymphatic EC identity genes Prox1, Vegfr3,and Lyve1. Using single-cell transcriptome data of human corpus cavernosum tissue, we found heterogeneity within pc-S endothelia and observed distinct transcriptional alterations related to inflammatory processes in hybrid ECs in erectile dysfunction associated with diabetes. Molecular, ultrastructural, and functional studies further established hybrid identity of pc-Ss in mouse, and revealed their morphological adaptations and ability to perform lymphatic-like function in draining high-molecular-weight tracers. Interestingly, we found that inhibition of the key lymphangiogenic growth factor VEGF-C did not block the development of pc-Ss in mice, distinguishing them from other lymphatic and hybrid vessels analyzed so far. Our findings provide a detailed molecular characterization of hybrid pc-Ss and pave the way for the identification of molecular targets for therapies in conditions of dysregulated penile vasculature, including erectile dysfunction.

GalR3 activation promotes adult neural stem cell survival in response to a diabetic milieu.

Type 2 diabetes impairs adult neurogenesis which could play a role in the CNS complications of this serious disease. The goal of this study was to determine the potential role of galanin in protecting adult neural stem cells (NSCs) from glucolipotoxicity and to analyze whether apoptosis and the unfolded protein response were involved in the galanin-mediated effect. We also studied the regulation of galanin and its receptor subtypes under diabetes in NSCs in vitro and in the subventricular zone (SVZ) in vivo. The viability of mouse SVZ-derived NSCs and the involvement of apoptosis (Bcl-2, cleaved caspase-3) and unfolded protein response [C/EBP homologous protein (CHOP) Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP), spliced X-box binding protein 1 (XBP1), c-Jun N-terminal kinases (JNK) phosphorylation] were assessed in the presence of glucolipotoxic conditions after 24 h. The effect of diabetes on the regulation of galanin and its receptor subtypes was assessed on NSCs in vitro and in SVZ tissues isolated from normal and type 2 diabetes ob/ob mice. We show increased NSC viability following galanin receptor (GalR)3 activation. This protective effect correlated with decreased apoptosis and CHOP levels. We also report how galanin and its receptors are regulated by diabetes in vitro and in vivo. This study shows GalR3-mediated neuroprotection, supporting a potential future therapeutic development, based on GalR3 activation, for the treatment of brain disorders.

Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation.

Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type-specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3caH1047R-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3caH1047R mice. Expression of pro-inflammatory genes, including monocyte/macrophage chemokine Ccl2, in Pik3caH1047R-iLECs was associated with recruitment of VEGF-C-producing macrophages. Macrophage depletion, CCL2 blockade, or anti-inflammatory COX-2 inhibition limited Pik3caH1047R-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk involving iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes.

Disruption of serine/threonine protein phosphatase 5 (PP5:PPP5c) in mice reveals a novel role for PP5 in the regulation of ultraviolet light-induced phosphorylation of serine/threonine protein kinase Chk1 (CHEK1).

PP5 is a ubiquitously expressed Ser/Thr protein phosphatase. High levels of PP5 have been observed in human cancers, and constitutive PP5 overexpression aids tumor progression in mouse models of tumor development. However, PP5 is highly conserved among species, and the roles of PP5 in normal tissues are not clear. Here, to help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. In marked contrast to the early embryonic lethality associated with the genetic disruption of other PPP family phosphatases (e.g. PP2A and PP4), intercrosses with mouse lines that ubiquitously express Cre recombinase starting early in development (e.g. MeuCre40 and ACTB-Cre) produced viable and fertile PP5-deficient mice. Phenotypic differences caused by the total disruption of PP5 were minor, suggesting that small molecule inhibitors of PP5 will not have widespread systemic toxicity. Examination of roles for PP5 in fibroblasts generated from PP5-deficient embryos (PP5(-/-) mouse embryonic fibroblasts) confirmed some known roles and identified new actions for PP5. PP5(-/-) mouse embryonic fibroblasts demonstrated increased sensitivity to UV light, hydroxyurea, and camptothecin, which are known activators of ATR (ataxia-telangiectasia and Rad3-related) kinase. Further study revealed a previously unrecognized role for PP5 downstream of ATR activation in a UV light-induced response. The genetic disruption of PP5 is associated with enhanced and prolonged phosphorylation of a single serine (Ser-345) on Chk1, increased phosphorylation of the p53 tumor suppressor protein (p53) at serine 18, and increased p53 protein levels. A comparable role for PP5 in the regulation of Chk1 phosphorylation was also observed in human cells.

Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells.

Pancreatic ß-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the ß-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary ß-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this ß-cell model.

Pituitary adenylate cyclase-activating polypeptide counteracts the impaired adult neural stem cell viability induced by palmitate.

Diabetes and obesity are characterized by hyperlipidemia and represent risk factors for premature neurological disorders. Diabetic/obese animals have impaired adult neurogenesis. We hypothesize that lipotoxicity leading to neurogenesis impairment plays a role in the development of neurological complications. If so, normalizing neurogenesis in diabetes/obesity could be therapeutically useful in counteracting neurological dysfunction. The goal of this study was to determine the potential of pituitary adenylate cyclase-activating polypeptide (PACAP) to protect adult neural stem cells (NSCs) from lipotoxicity and to study the expression of PACAP receptors in NSCs under lipotoxic conditions in vitro and in the subventricular zone in vivo. The viability of NSCs isolated from the adult mouse brain subventricular zone was assessed in the presence of a high-fat milieu, as mimicked by palmitate, which characterizes diabetic lipotoxicity. Regulation studies of PACAP receptors were performed by quantitative PCR on NSCs in vitro or on subventricular tissues isolated from obese ob/ob mice and their lean littermates. We show that palmitate impairs NSC viability by promoting lipoapoptosis. We also show that PACAP counteracts lipotoxicity via PAC-1 receptor activation. Studies on PACAP receptor expression revealed that PAC-1 and VPAC-2 are expressed by NSC in vitro and are upregulated by palmitate treatment and that PAC-1, VPAC-1, and VPAC-2 are expressed in the subventricular zone/striatum in vivo and are upregulated in ob/ob mice. The present study reveals a previously uncharacterized role of PACAP to protect NSC from lipotoxicity and suggests a potential therapeutic role for PACAP receptor agonists in the treatment of neurological complications in obesity and diabetes.

Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats.

Diabetes is a strong risk factor for premature and severe stroke. The GLP-1R (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto­Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 µg/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2­4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-1R agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions.

Cdh5-lineage-independent origin of dermal lymphatics shown by temporally restricted lineage tracing.

The developmental origins of lymphatic endothelial cells (LECs) have been under intense research after a century-long debate. Although previously thought to be of solely venous endothelial origin, additional sources of LECs were recently identified in multiple tissues in mice. Here, we investigated the regional differences in the origin(s) of the dermal lymphatic vasculature by lineage tracing using the pan-endothelial Cdh5-CreER T2 line. Tamoxifen-induced labeling of blood ECs at E9.5, before initiation of lymphatic development, traced most of the dermal LECs but with lower efficiency in the lumbar compared with the cervical skin. By contrast, when used at E9.5 but not at E11.5, 4-hydroxytamoxifen, the active metabolite of tamoxifen that provides a tighter window of Cre activity, revealed low labeling frequency of LECs, and lymphvasculogenic clusters in the lumbar skin in particular. Temporally restricted lineage tracing thus reveals contribution of LECs of Cdh5-lineage-independent origin to dermal lymphatic vasculature. Our results further highlight Cre induction strategy as a critical parameter in defining the temporal window for stage-specific lineage tracing during early developmental stages of rapid tissue differentiation.

Arachidonic acid fights palmitate: new insights into fatty acid toxicity in ß-cells.

Saturated fatty acids are toxic to pancreatic ß-cells. By inducing apoptosis, they contribute to a decrease in ß-cell mass, a hallmark of Type 2 diabetes. In the present issue of Clinical Science, Keane and co-workers show that the polyunsaturated fatty acid arachidonic acid protects the ß-cell against the toxic effects of palmitate. As Type 2 diabetes is characterized by subclinical inflammation, and arachidonic acid and metabolites thereof are produced during states of inflammation, it is possible that pancreatic ß-cells use arachidonic acid as a compound for self-protection.

Coadministration of sitagliptin or metformin has no major impact on the adverse metabolic outcomes induced by dexamethasone treatment in rats.

AIMS: Glucocorticoids (GC) in excess cause glucose intolerance and dyslipidemia due to their diabetogenic actions. Conceptually, antidiabetic drugs should attenuate these side effects. Thus, we evaluated whether the coadministration of metformin or sitagliptin (or both) with dexamethasone could attenuate GC-induced adverse effects on metabolism. MATERIALS AND METHODS: Adult male rats were treated for 5 consecutive days with dexamethasone (1 mg/kg, body mass (bm), intraperitoneally). Additional groups were coadministered with metformin (300 mg/kg, bm, by oral gavage (og)) or sitagliptin (20 mg/kg, bm, og) or with both compounds in combination. The day after the last treatments, rats were submitted to glucose tolerance tests, pyruvate tolerance test, and euthanized for biometric, biochemical, morphologic, and molecular analyses. KEY FINDINGS: Dexamethasone treatment resulted in reduced body mass and food intake, increased blood glucose and plasma insulin, dyslipidemia, glucose intolerance, pyruvate intolerance, and increased hepatic content of glycogen and fat. Sitagliptin coadministration improved glucose tolerance compared with the control group, an effect paralleled with higher levels of active GLP-1 during an oral GTT. Overall, sitagliptin or metformin coadministration did not prevent any of the dexamethasone-induced metabolic disturbances. SIGNIFICANCE: Coadministration of sitagliptin or metformin result in no major improvement of glucose and lipid metabolism altered by dexamethasone treatment in male adult rats.

Blockade of VEGF-C signaling inhibits lymphatic malformations driven by oncogenic PIK3CA mutation.

Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3CAH1047R mutation, resulting in constitutive activation of the p110α PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CAH1047R-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110α activation determining the LM subtype. In the postnatal vasculature, PIK3CAH1047R promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways.

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity.

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.

Lymph vessels are thin walled tubes that, similar to blood vessels, carry white blood cells, fluids and waste. Unlike veins and arteries, however, lymph vessels do not carry red blood cells and their main function is to remove excess fluid from tissues. The cells that line vessels in the body are called endothelial cells, and they are tightly linked together by proteins to control what goes into and comes out of the vessels. The chemical, physical and mechanical signals that control the junctions between endothelial cells are often the same in different vessel types, but their effects can vary. The endothelial cells of both blood and lymph vessels have two interacting proteins on their membrane known as EphrinB2 and its receptor, EphB4. When these two proteins interact, the EphB4 receptor becomes activated, which leads to changes in the junctions that link endothelial cells together. Frye et al. examined the role of EphrinB2 and EphB4 in the lymphatic system of mice. When either EphrinB2 or EphB4 are genetically removed in newborn or adult mice, lymph vessels become disrupted, but no significant effect is observed on blood vessels. The reason for the different responses in blood and lymph vessels is unknown. The results further showed that lymphatic endothelial cells need EphB4 and EphrinB2 to be constantly interacting to maintain the integrity of the lymph vessels. Further examination of human endothelial cells grown in the laboratory revealed that this constant signalling controls the internal protein scaffold that determines a cell's shape and integrity. Changes in the internal scaffold affect the organization of the junctions that link neighboring lymphatic endothelial cells together. The loss of signalling between EphrinB2 and EphB4 in lymph vessels reflects the increase in vessel leakage seen in response to bacterial infections and in some genetic conditions such as lymphoedema. Finding ways to control the signalling between these two proteins could help treat these conditions by developing drugs that improve endothelial cell integrity in lymph vessels.

Proteomics in diabetes research.

Both type 1 and type 2 diabetes mellitus are heterogeneous diseases with alterations in many genes and their products. Not all transcriptional alterations lead to protein changes, which makes it very important to, in conjunction with mRNA expression studies, also address changes in cellular protein levels. Various proteomic techniques are available for measuring many protein changes simultaneously. Many proteomic studies have been performed in the context of diabetes research, with the aims of both describing the healthy tissue and to unravel the complex pathophysiology behind the disease. In addition, effects on proteins induced by different treatments have also been investigated using proteomic approaches. In this paper the field of diabetes proteomics today will be reviewed. Findings from proteomic studies investigating pancreatic islets and beta-cells as well as serum, fat, skeletal muscle and liver are described.