Michele D ' Amico
email: michele.damico@unina2.it

Clara Di Filippo

Raffaele Marfella

Angela Maria Abbatecola

Franca Ferraraccio

Francesco Rossi

Giuseppe Paolisso

Michele D'amico

LONG-TERM inhibition of dipeptidyl peptidase-4 in Alzheimer's prone mice

Keywords: Alzheimer's, mice, dipeptidyl peptidase-4, sitagliptin, beta-amyloid, interleukin-1β

We tested here the impact of a long-term inhibition of dipeptidyl peptidase-4 (DPP-4) with sitagliptin on the deposition of amyloid beta within the brain and deficits in memory-related behavioral paradigms in a model of Alzheimer's disease (AD): double transgenic mice B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J. Mice began to receive sitagliptin at 7 months of age. Three different dose of sitagliptin (5, 10 20 mg/kg), were administered daily for 12 weeks by gastric gavage.

The treatments counteracted: i) the memory impairment in the contextual fear conditioning test; ii) increased the brain levels of GLP-1; iii) produced significant reductions of nitrosative stress and inflammation hallmarks within the brain, as well as iv) a significant diminution in the ultimate number and total area of βAPP and Aβ deposits. All these effects much more evident for the dose of 20 mg/kg sitagliptin. The long-term inhibition of the endogenous DPP-4 enzymes with sitagliptin can significantly delay some forms of AD pathology, including amyloid deposition, when administered early in the disease course of a transgenic mouse model of AD.

Introduction

AD is a complex neurodegenerative disorder, with aging, genetic and environmental factors contributing to its development and progression. The complexity of AD presents substantial challenges for the development of new therapeutic agents. AD is typified by pathological depositions of beta-amyloid peptides and neurofibrillary tangles within the diseased brain [START_REF] Jiang | The role of peroxisome proliferator-activated receptor-gamma (PPARgamma) in Alzheimer's disease: therapeutic implications[END_REF]. It has also been demonstrated to be associated with a significant microglia-mediated inflammatory component, dysregulated lipid homeostasis and regional deficits in glucose metabolism within the brain. AD also shares some common pathophysiologic hallmarks, such as amyloid beta (Abeta), phosphoralation of tau protein, and glycogen synthase kinase-3 with type 2 diabetes mellitus [START_REF] Li | Is Glucagon-like peptide-1, an agent treating diabetes, a new hope for Alzheimer's disease?[END_REF], and thus some drugs acting in diabetes to reduce the incidence of the adverse action of these mediators also promise to be useful in the prevention/reduction of Alzheimer's. This is the case for example of the incretin Glucagon-like peptide-1 (GLP-1). In patients with type 2 diabetes GLP-1 increases pancreatic islet beta-cell proliferation and glucosedependent insulin secretion; it lowers blood glucose and food intake. GLP-1 has cAMP coupled receptors within the brain of rodents and humans, thus, suggesting a central role for the endogenous peptide in the regulation of proper neuronal function as it is plasticity, cell survival, neurite outgrowth and protection against excitotoxic cell death and oxidative injury (Perry and Greig, 2002). Moreover, from experimental studies GLP-1 was shown to reduce endogenous levels of amyloid-beta peptide (Abeta) in mouse brain and to reduce levels of beta-amyloid precursor protein (betaAPP) in neurons (Perry and Greig, 2003) overall these data suggesting that GLP-1 peptide may beneficially affect a number of therapeutic targets associated with AD.

Unfortunately, GLP-1 peptide usually have short half-life in presence of functionally intact endogenous DPP-4 enzymes. These enzymes usually metabolize GLP-1 within minutes of release to inactive metabolites [START_REF] Drucker | Enhancing incretin action for the treatment of type 2 diabetes[END_REF][START_REF] Hansen | Glucagon-like peptide-1-(7-36) amide is transformed to glucagon-like peptide-1-(9-36)amide by dipeptidyl peptidase IV in the capillaries supplying the L cells of the porcine intestine[END_REF] resulting in very short half-life of intact hormone (about 2 minutes) [START_REF] Drucker | Enhancing incretin action for the treatment of type 2 diabetes[END_REF]Vilsboll et al., 2003) with the consequence that the small amounts of active GLP-1 that survive this initial cleavage act on receptor sites producing the proper responses of the peptide [START_REF] Drucker | The biology of incretin hormones[END_REF][START_REF] Meier | Clinical endocrinology and metabolism. Glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide[END_REF]. By blocking the activity of the endogenous DPP-4 enzymes one would stabilize the levels of the bioactive GLP-1 [START_REF] Brown | The burden of treatment failure in type 2 diabetes[END_REF][START_REF] Knecht | Diabetes care in the hospital: is there clinical inertia?[END_REF] and in theory its effects. Based on this contention, and although much remains to be elucidated with regards to the downstream signaling pathways involved in the properties of GLP-1, we have investigated here an alternative strategy to prevent/treat AD. We aimed to increase the bioactivity of endogenous GLP-1 with a chronic orally administered specific DPP-4 inhibitor, and investigate the effects of this continuous activation of the endogenous incretin axis on the deposition of Aß deposits within the brain and deficits in memory-related behavioural paradigms typical of AD.

Among the DPP-4 inhibitors we selected sitagliptin recently approved for the treatment of type 2 diabetes with a glucose-lowering effect best obtained after long-term treatment [START_REF] Lamont | Differential antidiabetic efficacy of incretin agonists versus DPP-4 inhibition in high fat fed mice[END_REF]. Sitagliptin (Januvia®, Merck Pharmaceuticals) is effective in lowering HbA1c, and fasting as well as postprandial glucose in monotherapy and in combination with other oral antidiabetic agents. It stimulates insulin secretion when hyperglycemia is present and inhibits glucagon secretion [START_REF] Gallwitz | Review of sitagliptin phosphate: a novel treatment for type 2 diabetes[END_REF]. Recent studies underline that the continuous administration of des-fluoro-sitagliptin in the food likely achieves optimal GLP-1 control by a potent and sustained 24-h inhibition of DPP-4 activity especially relative to the twice-daily administration of others DPP-4 inhibitors [START_REF] Lamont | Differential antidiabetic efficacy of incretin agonists versus DPP-4 inhibition in high fat fed mice[END_REF][START_REF] Larsen | Glucagon-like peptide-1 infusion must be maintained for 24 h/day to obtain acceptable glycemia in type 2 diabetic patients who are poorly controlled on sulphonylurea treatment[END_REF], Raun et.al., 2007,) with an end-point being the optimal stimulation of incretin axis and incretin activities.

Methods

Animals

The animals used were the double transgenic mice B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J from the Jackson Laboratory (Bar Harbor, USA). These transgenic mice express a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) both directed to CNS neurons. Both mutations are associated with early-onset (usually 6-7 months) Alzheimer's disease. By nine months of age, histological examination of brain tissue reveals numerous amyloid deposits within the hippocampus and cortex [START_REF] Jankowsky | Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase[END_REF] resembling those observed in the brains of patients with AD. The number of amyloid deposits increases dramatically between the ages of 10 and 12 months, with an accompanying profound inflammatory component being apparent. Mice (B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J, and lean littermates; n = 6 per group) of 7 months of age were used for experiments. They were fed with Purina 5002 rodent chow and drinking water. Sitagliptin was dissolved in normal saline for p.o. administration [START_REF] Beconi | Disposition of the dipeptidyl peptidase 4 inhibitor sitagliptin in rats and dogs[END_REF]. Vehicle (saline) and sitagliptin, at three different doses chosen in the therapeutic range of 11 mg/kg likely being 5, 10, 20 mg/kg, were daily administered by gastric gavage in a 100 microliter volume/10 g body weight. On completion of the study (after 12 weeks of drug treatment) mice were killed by cervical dislocation 2 h after their final morning sitagliptin or vehicle administration and the glucose assay repeated.

In a separate set of experiments exendin (9-39) was also tested. It was continuously administered through Alzet micropumps (Alza, Palo Alto, CA) delivering a rate of 2 pmol/kg/min [START_REF] Cani | Improvement of glucose tolerance and hepatic insulin sensitivity by oligofructose requires a functional glucagon-like peptide 1 receptor[END_REF] or vehicle (0.9% NaCl) for 12 weeks starting at 7 months of age. Animal care and handling was performed according to the local ethics committees.

Tissue preparation

At the time of sacrifice, the mice were transcardially perfused with heparinized sodium chloride (0.9%). The brains were removed and brain regions were dissected from one hemisphere. One hemisphere was placed into an acryl brain block lined up with the interaural line at 0 mm. Subsequently, coronal slices were cut to obtain cortical, hippocampal, cerebellar and brainstem sections, which were dissected, snap-frozen in liquid nitrogen and stored at -80°C until further analysis. The remaining brain hemisphere was either fixed in 4% paraformaldehyde followed by paraffin embedding according to standard protocols and sectioning for immunohistochemistry.

Immunohistochemistry

Serial sagittal sections of paraffin-embedded brain tissues were cut (7 µm thick, Leica microtome 2155 or Leica Cryostat CM3050S) and mounted (poly-L-lysine-coated slides, Histobond adhesion slides, Marienfeld, Germany). Retrieval of antigen sites, blocking of endogenous peroxidase activity and blocking of non-specific binding sites were performed according to standard protocols.

After washing in phosphate-buffered saline (PBS), sections were incubated overnight at 4°C with primary antibodies. The antibodies used were: (i) rabbit pAb against Aß1-42 (Biosource International, Inc., USA); and anti-β-APP (Santa Cruz); anti-IL-1β (Santa Cruz); anti-nitrotyrosine (Santa Cruz).

For image analysis of brain immunostaining, serial sagittal sections of one brain hemisphere were examined. β-APP, Aß1-42, immunostaining was evaluated on sagittal brain sections of six animals from each group. For each animal, antigens were detected in 10 parallel sections having a defined distance of 70 µm and showing both the hippocampus and brain cortex. In each section, the hippocampus and the frontal cortex were evaluated. The total stained area (real area) and integral staining density (sum of all individual optical densities of each pixel in the area being measured) were determined and given as percentage of stained surface per region. All images were acquired using a standard light and immunofluorescence microscope (Nikon, Eclipse E-800) connected to a digital camera (Sony, model DXC-9100P, Sony, Köln, Germany) and to a PC system with LUCIA imaging software (LUCIA 32G, version 4.11; Laboratory Imaging, Düsseldorf, Germany). For each animal, average values from all sections were determined.

Brain amyloid assay

The individual hemisphere was homogenized in 6.5 volumes of ice-cold buffer containing 20 mM Tris-HCl (pH 8.5) and a cocktail of proteinase inhibitors (Roche Mannheim, Germany), using a small Potter-type mechanical homogenizer. After centrifugation (135 000 g for 1 h at 4°C), a portion of the supernatant was centrifuged again (200 000 g for 2 h at 4°C) and soluble amyloid peptides were quantified by enzyme-linked immunosorbent assay (ELISA; IBL international, Hamburg, Germany; assay range 1.56-100 pg/ml).

In another set of experiments brain GLP-1 of B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice was assayed by using a commercially available ELISA kit (Linco Research, St. Charles, MO; 2pM sensitivity).

Statistical analysis

The data shown are the means ± S.D. Statistical comparisons for significance between mice with different treatments were performed using the Student's t test and ANOVA followed by the Tukey post hoc test.

Behavioural testings

Open-field test

This test was applied as previously described [START_REF] Onozuka | Nobiletin, a citrus flavonoid, improves memory impairment and Abeta pathology in a transgenic mouse model of Alzheimer's disease[END_REF]. Briefly, mice were placed into the corner of a wooden box 50 X 50 X 40 cm and allowed to freely explore for 10 min. The floor of the field was divided into 25 identical squares so that the ambulation of animals could be measured.

The ambulation of the mice was measured by counting the number of times that the animals crossed from one square to another. The scorer of the behavioral experiments was blind to treatment group.

Contextual fear conditioning

In the training session, mice were placed into a chamber with metal grids floor. Mice were allowed to freely explore for 2 min, and were given an electric shock (2 s, 0.7 mA) from a metal grid floor at the end of the 2 min. The 2 min/2 s shock paradigm was repeated for a total of two shocks. After the last shock, animals were allowed to explore the context for an additional 1 min prior to removal from the training chamber. The freezing time (seconds), defined as cessation of all but respiratory movement, was measured by observing the animals every 5 min. Baseline freezing was established for the first 2 min of the training session. Freezing time was also recorded during the last 3 min of the training session (training). In the test session performed 24 h after training, mice were placed into the conditioned chamber for 5 min and freezing time was measured manually (test).

Results

Inhibition of DPP-4 does not affect ambulation counts

Since B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice began to exhibit a number of amyloid deposits at 9 months of age [START_REF] Jankowsky | Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase[END_REF], 7 month-old transgenic mice were daily administered sitagliptin for 12 weeks. We carefully monitored the general health of the mice throughout the course of the treatment with sitagliptin and did not observe any adverse changes nor did we observe significant weight changes. After 12 weeks of the daily administration of sitagliptin we first examined the effects of sitagliptin on ambulation in the open field test. In this test, the number of crossed squares was similar between the vehicle-treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J and sitagliptin-treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J with no statistical significance between them (Figure 1), suggesting that treatment with sitagliptin did not affect general behaviour in B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice.

3.2 Inhibition of DPP-4 with sitagliptin counteracts the memory impairment in B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice To examine the effects of sitagliptin on learning and memory, we next used the contextual fear conditioning task, in which animals learn to associate a normally innocuous context with an aversive stimulus [START_REF] Kim | N-Methyl-D-aspartate receptor antagonist APV blocks acquisition but not expression of fear conditioning[END_REF][START_REF] Atkins | The MAPK cascade is required for mammalian associative learning[END_REF]. In the training session, there were no significant differences in the freezing time between the group vehicle-treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J and the sitagliptin-treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J. In contrast, the test session performed 24 h after training showed that treatment with sitagliptin (20 mg/kg) significantly improved the memory impairments in B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice (p<0.01) with the respect to vehicle-treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice, by increasing the freezing behavior of the animals against the aversive stimulus (Figure 2). The freezing time was 67% longer than the vehicle. Other lower doses of sitagliptin 5 and 10 mg/kg had no effect on this, with a calculated increase in percentage of freezing of 15% for the dose of 10 mg/kg (Figure 2).

Long-term inhibition DPP-4 increases brain endogenous levels of GLP-1

Figure 3 shows that B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice had increased levels of the incretin GLP-1 within the brain, following treatment with sitagliptin. This increasing effect of sitagliptin was maximally present after the dose of 20 mg/kg.

3.4

DPP-4 inhibition improves βAPP and Abeta pathology in B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice All mice were sacrificed after completion of the behavioral tests, and their brains were isolated and processed for neuropathological or biochemical evaluations. To determine the effects of the longterm administration of sitagliptin on amyloid beta deposition, we immunostained the brain sections from vehicle-and sitagliptin-treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice with antiamyloid beta antibody. At the age of 10 months, B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice expressed a considerable number of amyloid deposits throughout the cortex and hippocampus.

Since the contextual fear conditioning paradigm has been previously shown to be hippocampaldependent [START_REF] Logue | Hippocampal lesions cause learning deficits in inbred mice in the Morris water maze and conditioned-fear task[END_REF], we analyzed the βAPP and Abeta deposition in the hippocampus.

Notably, daily treatment with sitagliptin (10 and 20 mg/kg) for 12 weeks reduced both βAPP and Abeta deposition in the hippocampus of B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J (Figure 4 A and B). Our analysis demonstrated, as for example, that the treatment with sitagliptin resulted in about 60% decrease in the number of neurons within the area involved in the Abeta deposition as well as the amount of immunoreactivity compared with vehicle-treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J. In contrast exendin (9-39) treatment did not affect βAPP and βamyloid deposition in the brain (Figure 4A andB). It is noteworthy that exendin (9-39) is specific for the GLP-1 receptor and at the rate studied here, no nonspecific effect could be envisioned as previously described with higher rate [START_REF] Knauf | Brain glucagon-like peptide-1 increases insulin secretion and muscle insulin resistance to favor hepatic glycogen storage[END_REF].

We next analyzed quantity of Abeta with ELISA to investigate the effects of the DPP-4 inhibitor sitagliptin on Abeta levels in the brain. Administration of sitagliptin for 12 weeks significantly decreased levels of Abeta in the brain. As for example the levels of Abeta within the hippocampus was 12±1.6 pg/mg protein in vehicle-treated mice while it was 6.2±1.1 in sitagliptin-(20 mg/kg, p<0.01) treated B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J. Taken together with the results of immunohistochemical analysis, long-term treatment with sitagliptin reduced the amount of Abeta and inhibited formation of amyloid plaques in B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice.

3.5 Reduced expression of nitrosative stress and inflammation hallmarks within the brain of B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice following DPP-4 inhibition

As shown by the representative immunohistochemistriy of the brain, treatment of B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J mice with sitagliptin caused a substantial reduction of the inflammation and nitrosative stress within the brain associated with the reduced amyloid deposition described earlier. This effect was much more evident for the dose of 20 mg/kg sitagliptin (shown here by Figure 5). 10 mg had slight reductive action (data not shown) while 5 mg/kg had no significant effect.

Discussion

Epidemiological study suggests that 7.1% of all deaths for year were attributable to Alzheimer, placing it on a par with cerebrovascular disease as the third leading cause of death (Singer, 2005).

Recent study suggests that patients with AD had decreased survival compared with survival in the general U.S. population [START_REF] Larson | Survival after initial diagnosis of Alzheimer disease[END_REF]: men had a median survival of 4.2 years from their initial diagnosis, and women had a median survival of 5.7 years. Moreover, patients with AD were more likely to have cardiac left ventricular wall motion abnormalities, left ventricular hypertrophy, and reduced ejection fraction compared with patients affected by vascular dementia (Reitz et al., 2006).

Brain dysfunction in AD is characterized by loss of neocortical neurons [START_REF] Blennow | Alzheimer's disease[END_REF]) and focal amyloid deposits, which consist of the locally expressed amyloid ß protein (AßP). Formation of AßP by neurons is generally thought to be a prime trigger of the pathogenesis of AD. The generation of AßP is initiated by a protease that cleaves a larger precursor protein, the ß-amyloid precursor protein (ß-APP), at the N terminus side of the A peptide. ß-secretase or ß-site APPcleaving enzyme (BACE1) was cloned and identified as a transmembrane aspartyl protease [START_REF] Blennow | Alzheimer's disease[END_REF]. BACE1 deficiency precludes AßP formation in transgenic mice [START_REF] Mohajeri | Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology[END_REF] and does not cause or promote any neurological or phenotypic abnormalities.

Moreover, BACE1 inactivation rescues memory deficits in transgenic mice [START_REF] Lesnè | A specific amyloid-beta protein assembly in the brain impairs memory[END_REF], strongly supporting the importance of BACE1 as a therapeutic target in AD. However, recent objectives have been focused on a new strategy based on the reinforcement of the incretin axis by direct stimulation of it with administration of incretins or analogues or indirectly by blocking their degradation with dipeptidyl peptidase-4 inhibitors. The rationale of using these new classes of drugs in AD may resides in the fact that several studies nowadays have ascertained common pathophysiologic hallmarks, such as amyloid beta (Abeta) with type 2 diabetes mellitus [START_REF] Li | Is Glucagon-like peptide-1, an agent treating diabetes, a new hope for Alzheimer's disease?[END_REF], and have ascertained some diabetic drugs also useful in the prevention/reduction of Alzheimer's. This is the case for example of the incretin GLP-1 or DPP-4 inhibitors.

While many reports are present in literature on the use of the incretin GLP-1 or its analogues in AD, no study has investigated the second possibility that of use DPP-4 inhibitor in order to prevent amyloid deposits within the brain, thus preventing AD progression. DPP-4 is an enzyme that is localized vastly in the endothelium and can also be measured in the blood. DPP-4 cleaves peptides with an N-terminal alanine or proline amino acid residue [START_REF] Mentlein | Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum[END_REF]. In case of the incretin GLP-1 the two GLP-1 fragments resulting from DPP-4 activity are both biologically inactive; the fragment GLP-1(9-36) amide has even been described as having GLP-1 antagonistic properties in some studies [START_REF] Knudsen | Glucagon-like peptide-1-(9-36) amide is a major metabolite of glucagon-like peptide-1-(7-36) amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor[END_REF]. DPP-4 inhibition raises intact GLP-1 plasma concentrations to levels observed in the stimulated state after a meal. Further results in support come from mice deficient in DPP-4/CD26. These animals are resistant to STZ-induced diabetes and have elevated circulating plasma concentrations of intact GLP-1 and other incretin hormones [START_REF] Marguet | Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26[END_REF].

We report here that long-term treatment with the DPP-4 inhibitor sitagliptin increases the endogenous levels of GLP-1 and reduces the levels of amyloid-beta peptide (Abeta) and amyloid precursor protein (APP) in the brain in vivo. This result in agreement with the concept that GLP-1 reduces the levels of amyloid-beta peptide in the brain in vivo and reduces the levels of amyloid precursor protein (APP) in cultured neuronal cells, and with data that GLP-1 and exendin-4 (a GLP-1 analogue) protect cultured hippocampal neurons against death induced by Abeta and iron, an oxidative insult (Perry et al., 2003). Bearing in mind that endogenous GLP-1 possesses neurotropic properties [START_REF] Lamont | Differential antidiabetic efficacy of incretin agonists versus DPP-4 inhibition in high fat fed mice[END_REF] there is, therefore, a possibility that with the introduction of a DPP-4 inhibitor the successful management of GLP-1 release is maintained while still allowing GLP-1 activity to go unaltered and potentially act to positively reduce Aβ concentrations. In other word a DPP-4 inhibitor as it is sitagliptin would maintain the natural action of GLP-1 on the Aβ degradation and may add positive effects in term of reducing the cognitive impairment caused by AD.

On another note, we show here that DPP-4 inhibition is associated with a reduction of the inflammation and nitrosative stress within the cerebral structures. This is in agreement with many evidence that i) an inflammatory process in the central nervous system is believed to play an important role in the pathway leading to neuronal cell death [START_REF] Kubis | Alzheimer's disease: new prospects in therapy and applied experimental models[END_REF], and ii)

proinflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor are increased in AD [START_REF] Bryan | Expression of CD74 is increased in neurofibrillary tangles in Alzheimer's disease[END_REF].

Figure 1 .

 1 Figure 1. Effects of sitagliptin (5, 10 and 20 mg/kg) on ambulation counts in the open-field test in

Figure 2 .

 2 Figure 2. Effects of sitagliptin (5, 10 and 20 mg/kg) on freezing time (seconds) in the contextual

Figure 3 .

 3 Figure 3. Effects of sitagliptin (5, 10 and 20 mg/kg) on brain GLP-1 levels in B6.Cg-

Figure 4 .

 4 Figure 4. Representative hippocampal immunostaining (X200) for A) βAPP and for B) βamyloid

Figure 5 .

 5 Figure 5. Representative hippocampal immunostaining (X200) for A) nitrotyrosine and B) IL1-β

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Acknowledgements

This study was funded by the 2 nd University of Naples, Italy. There are no actual or potential conflicts of interest on this study.


V Christiaens

H R Lijnen

Angiogenesis and development of adipose tissue

Keywords: obesity, angiogenesis, adipocytes, endothelial cells, blood vessels

Obesity is a common disorder and related diseases, such as diabetes, atherosclerosis, hypertension, cardiovascular disease and cancer, are a major cause of mortality and morbidity in Western-type societies. Development of obesity is associated with substantial modulation of adipose tissue structure. The plasticity of the adipose tissue is reflected by its remarkable ability to expand or to reduce in size throughout adult lifespan. The expansion of adipose tissue is linked to the development of its vasculature. Indeed, adipogenesis is tightly associated with angiogenesis, as shown by the findings that adipose tissue explants trigger blood vessel formation, whereas in turn adipose tissue endothelial cells promote pre-adipocyte differentiation. Different components have been identified that play a role in adipose tissue associated angiogenesis. Modulation of angiogenesis may have the potential to impair adipose tissue development and thus may provide a novel therapeutic approach for prevention and treatment of obesity.

Introduction

Over the last decades obesity and its consequences worldwide have become a major health problem. Between 1976 and2002, the prevalence of overweight (body mass index (BMI) ≥ 25 kg/m 2 ) in the United States has increased from 46% to 66% of the population, and that of obesity (BMI ≥ 30 kg/m 2 ) from 15% to 31% [START_REF] Hedley | Prevalence of overweight and obesity among US children, adolescents, and adults 1999-2000[END_REF]. Excess weight increases the risk of multiple conditions, including hypertension, cardiovascular and cerebrovascular disease, type 2 diabetes, certain types of cancer, respiratory diseases, gallstones and osteoarthritis. Obesity is frequently associated with metabolic abnormalities such as impaired glucose tolerance, hyperinsulinemia, dyslipidemia with elevated triglyceride level, decreased high-density lipoprotein cholesterol concentration and increased proportion of small dense lipoparticles. This cluster of metabolic disturbances is called the metabolic syndrome, and represents known risk factors for cardiovascular disease. In addition, obesity negatively affects physical functioning, vitality, and general quality of life [START_REF] Kopelman | Obesity as a medical problem[END_REF][START_REF] Roth | The obesity pandemic; where have we been and where are we going?[END_REF]. Unlike most other tissues, adipose tissue continuously undergoes expansion and regression throughout adult life. Adipose tissue expansion requires the parallel growth of its capillary network. Therefore, adipogenesis (the formation of adipocytes) is tightly associated with angiogenesis (the formation of new blood vessels). We will review the pro-and anti-angiogenic components that have been demonstrated in adipose tissues, and discuss how modulation of angiogenesis may affect obesity.

Adipose tissue vasculature

A well-defined vascular system is present in adipose tissue, with every adipocyte surrounded by one or more capillaries. Fat cell development is characterized by the appearance of a number of fat cell clusters, or "primitive organs", which are vascular structures in the adipose tissue with few or no fat cells. During fetal development, arteriolar differentiation precedes adipocyte development and differentiation of blood vessel extracellular matrix (ECM) precedes differentiation of adipocyte ECM [START_REF] Hausman | Adipose tissue angiogenesis[END_REF]. Adipose tissue consists of mature adipocytes surrounded by a stromal-vascular cell fraction containing preadipocytes, endothelial cells, pericytes, fibroblasts, macrophages, and mesenchymal stem cells. Autocrine/paracrine or developmental relationships exist between capillaries/endothelial cells and preadipocytes. In vitro studies revealed that adipose tissue explants in fibrin or collagen gels trigger blood vessel formation [START_REF] Montesano | Vascular outgrowths from tissue explants embedded in fibrin or collagen gels: a simple in vitro model of angiogenesis[END_REF], and that in turn adipose tissue endothelial cells promote preadipocyte differentiation [START_REF] Varzaneh | Extracellular matrix components secreted by microvascular endothelial cells stimulate preadipocyte differentiation in vitro[END_REF]. Mature adipocytes in culture can dedifferentiate followed by differentiation into adipocytes or endothelial cells, suggesting that these are derived from a common lineage [START_REF] Planat-Benard | Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives[END_REF]. The growth of white adipose tissue (WAT) requires continuous remodeling of the vascular network, primarily of primitive capillary networks. Expansion of adipose tissue can be supported by both neovascularization (for adipocyte hyperplasia) and dilation and remodeling of existing capillaries (for adipocyte hypertrophy) [START_REF] Hausman | The histology of developing porcine adipose tissue[END_REF]. Brown adipose tissue (BAT) is mainly responsible for energy metabolism, and its function requires efficient blood perfusion to supply nutrients and oxygen and to export heat. BAT hyperplasia is critically dependent on angiogenesis, as it requires rapid A c c e p t e d M a n u s c r i p t 4 activation of mitosis in fat precursor cells and endothelial cells to develop capillaries [START_REF] Bukowiecki | Mechanism of enhanced lipolysis in adipose tissue of exercise-trained rats[END_REF]. Blood vessel density may not truly reflect angiogenic activity. Microvessel density is often considered a prognostic indicator for cancer. However, microvessel density does not reflect the angiogenic activity or angiogenic dependence of a tumor. The metabolic needs of cancer cells vary with the tissue of origin and change with tumor progression. Thus, the number of tumor cells that can be supported by a vessel varies, influencing in turn the vascular density [START_REF] Hlatky | Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us[END_REF]. In addition, capillary endothelium has important structural features such as microvascular endothelial cells surrounded by capillary fenestrations and transendothelial channels. Several soluble growth factors are found to regulate vascular fenestrations and permeability in adipose tissue. For example leptin increases the vascular permeability in adipose tissue and consequently, influences the microvessel density [START_REF] Cao | Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF[END_REF]. To take this into account, blood vessel density in adipose tissues can be normalized to the adipocyte density.

Regulation of adipose tissue related angiogenesis

Both WAT and BAT produce and secrete many different types of proangiogenic factors such as vascular endothelial growth factor (VEGF)-A and hepatocyte growth factor (HGF), the two key angiogenic factors produced by adipocytes. Other adipose-tissue derived factors with proangiogenic effects include VEGF-B, VEGF-C, placental growth factor (PlGF), fibroblast growth factor (FGF)-2, SPARC/osteonectin, angiopoietin (Ang)-1, Ang-2, leptin, platelet derived growth factor (PDGF)-, transforming growth factor (TGF)-, tumor necrosis factor (TNF)-, tissue factor (TF), matrix metalloproteinases (MMPs), plasminogen activators and cathepsins. Adipose tissue also produces endogenous antiangiogenic factors, such as adiponectin, thrombospondin (TSP)-1, TSP-2, ADAM and ADAMTS family members. Thus, the regulation of angiogenesis in adipose tissue may depend on the local balance between proangiogenic and antiangiogenic factors. The angiogenic potential of the main pro-or anti-angiogenic components is sometimes context dependent and different in different fat pads.

Vascular endothelial growth factors and placental growth factor

VEGF-A (17-23 kDa) is a major angiogenic factor that stimulates proliferation and migration of endothelial cells. Three forms of VEGF-A are produced in the mouse as a result of alternative splicing (VEGF-A121, VEGF-A165 and VEGF-A189). Several studies indicate that VEGF-A stimulates both physiological and pathological angiogenesis by signaling through vascular endothelial growth factor receptor (VEGFR)-2 in a strict dose-dependent manner. Loss of a single VEGF-A allele causes embryonic vascular defects, while reduction of VEGF-A levels by only 25% impairs spinal cord perfusion, resulting in motor neuron degeneration reminiscent of amyotrophic lateral sclerosis [START_REF] Carmeliet | Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[END_REF][START_REF] Ferrara | Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[END_REF][START_REF] Oosthuyse | Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration[END_REF]. VEGF-A is believed to be responsible for most of adipose tissue's angiogenic capacity [START_REF] Zhang | Vascular endothelial growth factor is the major angiogenic factor in omentum: mechanism of the omentum-mediated angiogenesis[END_REF]. VEGF-B (21 kDa) is 43% identical to VEGF-A165; it also promotes angiogenesis and is implicated in ECM degradation via regulation of plasminogen activation [START_REF] Olofsson | Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells[END_REF]. VEGF-C (23 kDa) displays 30% homology with VEGF-A165 and plays an important role both in angiogenesis and lymphangiogenesis [START_REF] Joukov | A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases[END_REF][START_REF] Karkkainen | Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins[END_REF]. VEGF-D (22 kDa) is 48% identical to VEGF-C and also A c c e p t e d M a n u s c r i p t promotes the growth of lymphatic vessels [START_REF] Stacker | VEGF-D promotes the metastatic spread of tumor cells via the lymphatics[END_REF]. VEGF is expressed by both stromavascular fraction cells and mature adipocytes, as described in both animals and humans [START_REF] Ledoux | Angiogenesis associated with visceral and subcutaneous adipose tissue in severe human obesity[END_REF]. PlGF, a 25 kDa homologue of VEGF-A (53% sequence identity with VEGF-A165), enhances angiogenesis, but only in pathological conditions. Loss of PlGF impairs angiogenesis in the ischemic retina, limb, and heart, in wounded skin and in tumors, without affecting physiological angiogenesis [START_REF] Carmeliet | Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions[END_REF]. PlGF is expressed in murine adipose tissue, both in adipocytes and in the stromal-vascular cell fraction [START_REF] Voros | Modulation of angiogenesis during adipose tissue development in murine models of obesity[END_REF].

VEGF and PlGF receptors

The members of the VEGF family bind to transmembrane tyrosine kinase receptors (VEGFR-1 (206 kDa), VEGFR-2 (218 kDa) and VEGFR-3 (150 kDa)). VEGF-A interacts with both VEGFR-1 and VEGFR-2, whereas VEGF-B and PlGF bind to VEGFR-1. VEGF-C and VEGF-D activate VEGFR-3, but VEGF-C can also bind to VEGFR-2. VEGFR-1 and VEGFR-2 mediate angiogenesis, whereas VEGFR-3 is involved mainly in lymphangiogenesis [START_REF] Veikkola | Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice[END_REF]. VEGF-A165, PlGF and VEGF-B also bind to another transmembrane receptor, neuropilin-1 (Np-1). Inactivation of the Np-1 gene in mice causes disturbances in development of the vascular and nervous system [START_REF] Kawasaki | A requirement for neuropilin-1 in embryonic vessel formation[END_REF].

Hepatocyte growth factor, fibroblast growth factor 2 and osteonectin

HGF is a mesenchyme-derived multifunctional molecule that elicits mitogenic and morphogenic activities in developmental as well as in many pathophysiological processes [START_REF] Weidmer | Molecular characteristics of HGF-SF and its role in cell motility and invasion[END_REF][START_REF] Jeffers | Hepatocyte growth factor/scatter factor-Met signaling in tumorigenicity and invasion/metastasis[END_REF]. Its receptor has been identified as the c-Met tyrosine kinase, a product of the proto-oncogene c-met. Both HGF and c-Met play a critical role in the invasive growth of tumor cells, a hallmark of metastatic cancers. HGF secreted by cultured 3T3-L1 adipocytes promotes tube formation of human umbilical vascular endothelial cells (HUVECs) in vitro [START_REF] Saiki | Hepatocyte growth factor secreted by cultured adipocytes promotes tube formation of vascular endothelial cells in vitro[END_REF]. FGF-2 (25 kDa) is a potent stimulator of differentiation, migration and proliferation of endothelial cells, and enhances adipocyte differentiation in vivo [START_REF] Kawaguchi | De novo adipogenesis in mice at the site of injection of basement membrane and basic fibroblast growth factor[END_REF]. During angiogenesis, FGF-2 stimulates the synthesis of proteinases such as collagenase and urokinase-type plasminogen activator (uPA), and of integrins to form new capillary cord structures [START_REF] Okamura | Endogenous basic fibroblast growth factor-dependent induction of collagenase and interleukin-6 in tumor necrosis factor-treated human microvascular endothelial cells[END_REF][START_REF] Tienari | The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells[END_REF]. SPARC/osteonectin is a matricellular protein that is thought to modulate cell-matrix interaction. It binds to VEGF-A, impairs VEGFR-1 activation, and inhibits FGF-2, resulting in inhibition of endothelial cell proliferation [START_REF] Bornstein | Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin-1[END_REF]. Osteonectin is produced by adipose tissue and its expression is upregulated in obesity (Tartare-Deckert et al., 2001). Osteonectin deficient mice on high fat diet (HFD) develop larger fat pads as compared to wild-type mice [START_REF] Bradshaw | SPARC-null mice exhibit increased adipocity without significant differences in overall body weight[END_REF].

The angiopoietins, TIE-1 and TIE-2

Another signaling system contributing to maintenance, growth and stabilization of blood vessels involves the tyrosine kinase (T) with Ig (I) and epidermal (E) growth factor (EGF) homology domains (TIE)-1 and -2 receptors (140-145 kDa). TIE-2 binds the angiopoietins (Ang-1 and Ang-2), whereas the ligands of TIE-1 are less well defined.

A c c e p t e d M a n u s c r i p t 6 Unlike Ang-2, which activates TIE-2 on some cells but blocks it on others [START_REF] Davis | Angiopoietins have distinct modular domains essential for receptor binding, dimerization and superclustering[END_REF], Ang-1 consistently activates TIE-2 [START_REF] Davis | Isolation of angiopoietin-1, a ligand for the TIE-2 receptor, by secretion-trap expression cloning[END_REF]. The role of Ang-1 in vascularization is pleiotropic and context-dependent. Ang-1 tightens vessels by affecting junctional molecules [START_REF] Thurston | Angiopoietin-1 protects the adult vasculature against plasma leakage[END_REF], by promoting the interaction between endothelial cells and mural cells and by recruiting pericytes [START_REF] Carlson | Direct cell adhesion to the angiopoietins mediated by integrins[END_REF]. Ang-2 may stimulate vessel growth by loosening endothelial-peri-endothelial cell interactions and degrading the ECM [START_REF] Morange | Influence of t-PA and u-PA on adipose tissue development in a murine model of diet-induced obesity[END_REF]. TIE-1 and TIE-2 are expressed by endothelial cells in murine adipose tissues and their expression increases with fat pad development. However, their role in adipose tissue associated angiogenesis has not been clearly established [START_REF] Neels | Angiogenesis in an in vivo model of adipose tissue development[END_REF].

Leptin

Leptin (16 kDa), a satiety hormone produced by mature adipocytes, promotes migration of endothelial cells. Interaction of leptin with its receptor on endothelial cells leads to activation of the Stat3 pathway and enhancement of its DNA binding activity [START_REF] Sierra-Honigmann | Biological action of leptin as an angiogenic factor[END_REF]. Besides a direct proangiogenic activity, leptin upregulates VEGF expression via activation of the Jak/Stat3 signaling pathway [START_REF] Suganami | Leptin stimulates ischemia-induced retinal neovascularization: possible role of vascular endothelial growth factor expressed in retinal endothelial cells[END_REF]. Similar to VEGF-A, leptin induces the formation of fenestrated capillaries, as confirmed by the absence of fenestrations in leptin deficient ob/ob mice. Leptin has a synergistic effect on stimulation by VEGF and FGF-2 [START_REF] Cao | Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF[END_REF].

Adiponectin

Adiponectin is an abundant circulating plasma protein secreted by mature adipocytes that mediates beneficial effects in the vasculature. Circulating levels of adiponectin are decreased in individuals with obesity and type 2 diabetes, suggesting a potential role for adiponectin in vascular diseases that frequently accompany these disorders [START_REF] Matsuzawa | Therapy insight: adipocytokines in metabolic syndrome and related cardiovascular disease[END_REF]. Pro-as well as anti-angiogenic effects of adiponectin have been reported. Adiponectin inhibited endothelial cell migration and proliferation in vitro and neoangiogenesis in vivo in the chick chorioallantoic membrane (CAM) and cornea assays, and it reduced angiogenesis and induced apoptosis in tumors, obtained by implanting T241 fibrosarcoma cells in mice [START_REF] Bråkenhielm | Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis[END_REF]. Adiponectin suppressed VEGF-induced migration of human coronary artery endothelial cells (HCAECs) via cAMP/PKA-dependent signaling [START_REF] Mahadev | Adiponectin inhibits vascular endothelial growth factor-induced migration of human coronary artery endothelial cells[END_REF]. However, adiponectin has also been reported to activate adenosine monophosphate kinase in endothelial cells, leading to enhanced in vivo angiogenesis in murine Matrigel plug and rabbit cornea assays and to inhibition of caspase 3-mediated apoptosis in HUVECs cultured in vitro [START_REF] Kobayashi | Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin[END_REF][START_REF] Ouchi | Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells[END_REF].

Growth factors and cytokines

PDGF was originally purified from platelets; however, it has since been found in many other cell types including fibroblasts, keratinocytes, myoblasts, astrocytes, epithelial cells, and macrophages [START_REF] Heldin | Mechanism of action and in vivo role of plateletderived growth factor[END_REF]. A recent study shows that macrophage-derived PDGF is involved in the stimulation of angiogenesis during expansion of adipose tissue [START_REF] Pang | Macrophage infiltration into adipose tissue may promote angiogenesis for adipose tissue remodeling in obesity[END_REF]. TGF- is a multifunctional cytokine that is produced by a variety of cells and is capable of regulating the growth and differentiation of many cell types. It is implicated in a A c c e p t e d M a n u s c r i p t number of biological processes including cell adhesion and migration, extracellular matrix production, tissue remodeling and wound repair [START_REF] Mccartney-Francis | Transforming growth factor beta: A matter of life and death[END_REF]. TGF- mRNA levels are increased in both the mature adipocyte and the stromal/vascular cell fractions of the adipose tissues from obese mice. The augmented expression of TGF- in the obese adipose tissue may increase adipocyte precursor cell proliferation, thereby contributing to the excessive cellularity of the fat depots associated with the obese phenotype [START_REF] Samad | Elevated expression of transforming growth factor- in adipose tissue from obese mice[END_REF]. TNF- is a multipotential cytokine with several immunological functions. It was the first product secreted from adipose tissue to be proposed as a molecular link between obesity and insulin resistance. In humans, adipose tissue TNF- mRNA correlates with body mass index, percentage of body fat and hyperinsulinaemia [START_REF] Katsuki | Serum levels of tumor necrosis factor-alpha are increased in obese patients with noninsulin-dependent diabetes mellitus[END_REF]. TNF- promotes endothelial cell tube formation in vitro and inhibits endothelial cell proliferation [START_REF] Papetti | Mechanisms of normal and tumor-derived angiogenesis[END_REF]. A functional role of TNF- in adipogenesis and insulin resistance has been studied in the genetically obese and diabetic ob/ob mice [START_REF] Uysal | Protection from obesity-insulin resistance in mice lacking TNF- function[END_REF]. TNF- deficient ob/ob mice showed lower levels of circulating free fatty acids, and were protected from the obesity-related reduction in the insulin receptor signaling in muscle and fat tissues. Interfering with TNF- signaling through null mutations in TNF receptors p55 and p75 also resulted in a significant protection from the insulin resistance associated with the ob/ob phenotype [START_REF] Uysal | Protection from obesity-insulin resistance in mice lacking TNF- function[END_REF].

Tissue factor

TF is the major cellular initiator of the coagulation cascade and serves as a cell-surface receptor for the activation of factor VII [START_REF] Camerer | Cell biology of tissue factor, the principal initiator of blood coagulation[END_REF]. TF is expressed by both mature adipocytes and stromal/vascular cell fractrions in murine adipose tissue and increased levels of TF in obesity promote the development of a hypercoagulable state and thus may contribute to the cardiovascular complications associated with obesity [START_REF] Samad | Tissue factor gene expression in the adipose tissues of obese mice[END_REF]. TF is involved in metastasis and indirectly in tumor growth via angiogenesis, as tumor cells transfected to overexpress TF expressed high levels of VEGF and reduced amounts of thrombospondin [START_REF] Zhang | Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice[END_REF].

Proteolytic systems

Initiation of angiogenesis requires vessel destabilization, which involves weakening of the intercellular contacts between endothelial cells, destruction of the basement membrane, and local proteolysis of matrix proteins to allow the endothelial cells to migrate and produce new capillaries [START_REF] Lamalice | Endothelial cell migration during angiogenesis[END_REF]. Proteases of at least three classes (serine, cysteine, and metallo-), including MMPs, members of the urokinase/ plasmin(ogen) system and cysteine cathepsins, play crucial roles in the remodeling of the ECM and therefore in angiogenesis. Proteases participate in the angiogenic process by generating both pro-and anti-angiogenic factors from ECM proteins, and by processing growth factors and receptors [START_REF] Van Hinsbergh | Pericellular proteases in angiogenesis and vasculogenesis[END_REF]. The MMPs belong to a family of over 25 neutral endopeptidases that play a role in ECM proteolysis. The vascular localization of MMPs may allow their spatial role in the degradation of subendothelial basement membrane, which is a crucial step in angiogenesis and atherosclerosis. MMPs promote angiogenesis by regulating endothelial cell attachment, proliferation and migration, either directly or by release of growth factors A c c e p t e d M a n u s c r i p t sequestered in the ECM [START_REF] Stetler-Stevenson | Matrix metalloproteinase in angiogenesis: a moving target for therapeutic intervention[END_REF]. Many MMPs are expressed by mature adipocytes and stromal-vascular cells in a depot-dependent manner [START_REF] Maquoi | Modulation of adipose tissue expression of murine matrix metalloproteinases and their tissue inhibitors with obesity[END_REF]. The ADAM (A Disintegrin And Metalloproteinase) family comprises proteins containing disintegrin-like and metalloproteinase-like domains [START_REF] Wolfsberg | ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloproteinase domain[END_REF]. The ADAMTS family includes a subset of ADAM proteins that contain a thrombospondin motif [START_REF] Kaushal | The new kids on the block: ADAMTSs, potentially multifunctional metalloproteinases of the ADAM family[END_REF]. Several ADAM and ADAMTS family members are expressed in adipose tissue and during differentiation of preadipocytes [START_REF] Voros | Differential expression of plasminogen activator inhibitor-1, tumor necrosis factor-, TNF- converting enzyme and ADAMTS family members in murine fat territories[END_REF]. ADAMTS-1 and ADAMTS-8 can inhibit VEGF-induced angiogenesis and suppress FGF-2-induced vascularization [START_REF] Vázquez | METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity[END_REF]. They both mediate a greater antiangiogenic response than either TSP-1 or endostatin, with ADAMTS-1 showing a greater inhibitory capacity than ADAMTS-8. The antiangiogenic activity of ADAMTS-1 and -8 is mediated through their TS motifs. Interestingly, ADAMTS-1 deficient mice are exceptionally lean, their volume of epididymal fat is significantly smaller than in wild-type mice and the formation of capillaries in the adrenal gland is drastically impaired [START_REF] Luque | ADAMTS1/METH1 inhibits endothelial cell proliferation by direct binding and sequestration of VEGF 165[END_REF]. The fibrinolytic system comprises an inactive proenzyme, plasminogen, that can be converted to the active enzyme, plasmin, that degrades fibrin into soluble fibrindegradation products. Two immunologically distinct plasminogen activators have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). u-PA binds to a specific cellular receptor (u-PAR or CD87) resulting in enhanced activation of cell-bound plasminogen. Inhibition of the fibrinolytic system may occur either at the level of plasmin by  2 -antiplasmin, or at the level of the plasminogen activators, mainly by plasminogen activator-inhibitor-1 (PAI-1) [START_REF] Lijnen | Molecular and cellular basis of fibrinolysis[END_REF]. The fibrinolytic system participates in diverse physiologic processes, including blood coagulation, tissue growth and remodeling, wound healing, angiogenesis and embryogenesis. Especially the involvement of uPA and uPAR in angiogenesis is well established. uPA stimulates endothelial cell tube formation on a fibrin matrix, whereas an antibody blocking urokinase binding to uPAR and to soluble uPAR inhibits the effects of uPA. Urokinase and uPA-generated plasmin can activate and release latent MMPs and elastase, as well as growth factors involved in angiogenesis, especially VEGF, bFGF, HGF, TGF, and PDGF [START_REF] Tkachuk | Regulation of arterial remodeling and angiogenesis by urokinase-type plasminogen activator[END_REF]. Several nutritionally induced obesity models in transgenic mice have been used to study the role of the fibrinolytic system in the development of obesity. t-PA-deficient mice, kept on high-fat diet, had higher body weight and adipose tissue mass than wild-type controls [START_REF] Morange | Influence of t-PA and u-PA on adipose tissue development in a murine model of diet-induced obesity[END_REF]. There was an increase in the number of endothelial cells and stromal cells in the fat tissues, suggesting that targeted inactivation of t-PA increases angiogenesis in adipose tissue. Deficiency in u-PA, in contrast, had no effect on nutritionally induced obesity [START_REF] Morange | Influence of t-PA and u-PA on adipose tissue development in a murine model of diet-induced obesity[END_REF]. Mice deficient in plasminogen showed reduced fat accumulation associated with reduced differentiation of stromal cells [START_REF] Hoover-Plow | In vivo plasminogen deficiency reduces fat accumulation[END_REF]. Deficiency of 2-antiplasmin had no significant effect on adipose tissue development in mice [START_REF] Lijnen | Deficiency of 2-antiplasmin does not affect murine adipose tissue development[END_REF]. Some studies with transgenic mice reported that PAI-1 deficiency has little or no effect on adipose tissue development [START_REF] Morange | Influence of PAI-1 on adipose tissue growth and metabolic parameters in a murine model of diet-induced obesity[END_REF][START_REF] Lijnen | Effect of plasminogen activator inhibitor-1 deficiency on nutritionally-induced obesity in mice[END_REF], whereas others found impaired development [START_REF] Ma | Prevention of obesity and insulin resistance in mice lacking plasminogen activator inhibitor-1[END_REF]. Disruption of the PAI-1 gene in ob/ob mice resulted in reduced adiposity and improvement of the metabolic profile [START_REF] Schäfer | Disruption of the plasminogen activator inhibitor-1 gene reduces the adiposity and improves the metabolic profile of genetically obese and diabetic ob/ob mice[END_REF].

A c c e p t e d M a n u s c r i p t

Cathepsin B, a lysosomal cysteine protease, regulates both pro-and anti-angiogenic factors suggesting that it may be a contributor to the "angiogenic switch" of endothelial cells [START_REF] Im | Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells[END_REF]. Lack of cathepsin B reduces angiogenesis in a mouse model of pancreatic islet cell carcinogenesis (RIP1-Tag2) crossed with cathepsin B-null mice [START_REF] Gocheva | Distinct roles for cysteine cathepsin genes in multistage tumorigenesis[END_REF]. Cathepsin B has also been shown to exert antiangiogenic effects through its ability to cleave collagen XVIII to generate endostatin, an antiangiogenic factor present in caveolae of endothelial cells. Active cathepsin B is also associated with caveolae and degradation and remodeling of the ECM during endothelial cell migration and tube formation [START_REF] Cavallo-Medved | Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation[END_REF]. Cathepsin B is found to be secreted by human adipose tissue [START_REF] Alvarez-Llamas | Characterization of the human visceral adipose tissue secretome[END_REF]. Overall, the balance of protease activity and the interaction of proteases with the ECM contribute to regulation of the angiogenic process.

Thrombospondins

TSP-1 and TSP-2 are components of the ECM in remodeling tissues and, like other matricellular proteins, bind to matrix proteins and cell-surface receptors, including proteoglycans, non-integrin and integrin receptors. TSPs can inhibit angiogenesis in several different ways (migration, proliferation, tube formation, apoptosis of endothelial cells) in vitro [START_REF] Armstrong | Thrombospondins 1 and 2 function as inhibitors of angiogenesis[END_REF]. Despite its pleiotropic biological role, mice deficient in TSP-1 are viable and exhibit only subtle abnormalities in development, although they develop pneumonia and show delayed organization and neovascularization of skin wounds [START_REF] Agah | The lack of thrombospondin-1 (TSP1) dictates the course of wound healing in double-TSP1/TSP2-null mice[END_REF]. In contrast, TSP-2 deficient mice display a pleiotropic phenotype that includes fragile skin, which is associated with abnormal collagen fibrillogenesis, increased vascularity primarily in reponse to injury, increased cortical bone density, and a bleeding diathesis [START_REF] Kyriakides | Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, an a bleeding diathesis[END_REF]. Both TSP-1 and TSP-2 are found to be secreted by human adipose tissue [START_REF] Alvarez-Llamas | Characterization of the human visceral adipose tissue secretome[END_REF].

Modulation of angiogenesis in adipose tissue

Modulation of development of the vascular network in adipose tissue may constitute a strategy to affect obesity. Therefore, it is important to obtain information on expression and functional role of pro-and anti-angiogenic components in in vitro models of adipogenesis and in in vivo models of obesity.

Expression of angiogenic factors during adipose tissue development

During differentiation of 3T3-F442A preadipocytes into mature adipocytes mRNAs for Ang-1, the VEGF-A isoforms and PlGF are upregulated; FGF-2 and Np-1 expression is transiently upregulated, whereas the expression of Ang-2 is high in confluent cultures but decreases during maturation. TSP-1 and TSP-2 expression are downregulated in the early phase of differentiation but increase again in later phases. VEGF-B, VEGF-C and VEGFR-1 mRNA levels are less modulated during differentiation. TIE-1, TIE-2, VEGFR-2 and VEGFR-3 are not expressed at detectable levels [START_REF] Voros | Modulation of angiogenesis during adipose tissue development in murine models of obesity[END_REF]. Development of vasculature and mRNA expression of pro-or anti-angiogenic factors were also studied during adipose tissue development in nutritionally induced (HFD) or genetically determined (ob/ob) murine obesity models [START_REF] Voros | Modulation of angiogenesis during adipose tissue development in murine models of obesity[END_REF]. Ob/ob mice and C57Bl/6 mice on HFD had significantly larger subcutaneous (SC) and gonadal (GON) fat pads, accompanied by significantly higher blood content, increased total blood A c c e p t e d M a n u s c r i p t 10 vessel volume and higher number of proliferating endothelial cells, as compared to lean controls. Ang-1 expression was downregulated, whereas TSP-1 was upregulated in developing adipose tissue in both obesity models. Despite this upregulation, TSP-1 deficiency in mice did not significantly affect adipose tissue development [START_REF] Voros | Deficiency of thrombospondin-1 in mice does not affect adipose tissue development[END_REF]; its role in adipose tissue associated angiogenesis thus remains unclear. Ang-1 mRNA levels correlated negatively, and TSP-1 mRNA levels correlated positively with adipose tissue weight. PlGF and Ang-2 expression were increased in SC adipose tissue of ob/ob mice and TSP-2 was increased in both their SC and GON fat pads. mRNA levels of VEGF-A isoforms, VEGF-B, VEGF-C, VEGFR-1, -2 and -3 and Np-1 were not markedly modulated by obesity [START_REF] Voros | Modulation of angiogenesis during adipose tissue development in murine models of obesity[END_REF]. Several angiogenic factors, like VEGF-C, VEGF-D, sVEGFr2 angiopoietin-2, angiogenin and the angiogenesis inhibitor endostatin are significantly elevated in either overweight or obese subjects compared to lean controls. Although it remains to be proven, it is likely that adipose tissue itself contributes to the elevated circulating levels of these factors. Therefore, adipose tissue may contribute to increased risk of metastatic disease in obese subjects with cancer [START_REF] Silha | Angiogenic factors are elevated in overweight and obese individuals[END_REF].

Animal models of genetic modification of angiogenic factors

Nutritionally induced obesity models in transgenic mice were used to study the role of some angiogenic factors in adipose tissue development.

To establish a functional role in obesity, PlGF deficient (PlGF -/-) and wild type mice with the same genetic background were kept on HFD for 15 weeks. PlGF -/-mice had a significantly lower body weight and less total SC plus GON adipose tissue. Blood vessel size was lower in GON adipose tissue of PlGF -/-mice, and blood vessel density, normalized to adipocyte number, was significantly lower in SC adipose tissue of PlGF -/- mice. These differences were not observed for PlGF -/-and wild type mice kept on normal chow. These observations are in line with the emerging concept that PlGF deficiency has little or no effect on angiogenesis under normal (feeding) conditions, but is associated with impaired angiogenesis under stress conditions (HFD) during the early stages of adipose tissue formation [START_REF] Lijnen | Impaired adipose tissue development in mice with inactivation of placental growth factor function[END_REF]. The contribution of HGF to adipose tissue angiogenesis was examined using the 3T3-F442A in vivo fat pad formation model [START_REF] Green | Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line[END_REF]. Silencing of HGF in 3T3-F442A preadipocytes, before injection under the skin of nude mice, impaired the ability of the developing fat pads to recruit endothelial cells for neovascularization. On the other hand, excess HGF accelerated endothelial cell migration in the fat pad [START_REF] Bell | A central role for hepatocyte growth factor in adipose tissue angiogenesis[END_REF]. Several findings suggest that lymphangiogenesis may also play an important role in development of adipose tissue. The observation that mice lacking Prox1, a gene required for the formation of lymphatic endothelial cells, display adult-onset obesity indicates a link between lymph fluid and fat deposition [START_REF] Harvey | Lymphatic vascular defects promoted by Prox1 haploinsufficiency cause adult-onset obesity[END_REF]. Adipogenesis and fat deposition are known to occur efficiently around lymph nodes, where the adipocytes provide energy for local lymphoid metabolic needs [START_REF] Schneider | Lymph makes you fat[END_REF]. VEGF-D is mitogenic for endothelial cells and has angiogenic as well as lymphangiogenic potential in vivo [START_REF] Saharinen | Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation[END_REF]. However, a nutritionally induced obesity model in VEGF-D deficient mice did not reveal an important role of VEGF-D in (lymph) angiogenesis or in adipose tissue development [START_REF] Lijnen | Deficiency of vascular endothelial growth factor-D does not affect murine adipose tissue development[END_REF]. These results do, A c c e p t e d M a n u s c r i p t 11 however, not allow to conclude that lymphangiogenesis would not play a role in adipose tissue development.

Pharmacologic inhibition of angiogenesis impairs adipose tissue development

3T3-F442A preadipocytes injected into severe combined immunodeficient (SCID) mice induce angiogenesis and differentiate into adipocytes [START_REF] Mandrup | Obese gene expression at in vivo levels by fat pads derived from s.c. implanted 3T3-F442A preadipocytes[END_REF]. It was reported that neovasculature in the new adipose tissue originates by sprouting from larger hostderived blood vessels that run parallel to peripheral nerves and that endothelial progenitor cells do not play an important role in this process [START_REF] Neels | Angiogenesis in an in vivo model of adipose tissue development[END_REF]. Adipogenesis and de novo fat pad formation in this model can be impaired by inhibition of PPAR- or VEGFR-2 [START_REF] Fukumura | Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis[END_REF] and by administration of a PlGF neutralizing monoclonal antibody [START_REF] Lijnen | Impaired adipose tissue development in mice with inactivation of placental growth factor function[END_REF]. Furthermore, adipose tissue growth in mice can be impaired with angiogenesis inhibitors such as TNP-470 (a synthetic analog of fumagillin, that selectively inhibits endothelial cell growth by suppression of methionine aminopeptidase), angiostatin (a plasminogen fragment containing kringles 1-4) and endostatin (a COOH-terminal fragment of collagen XVIII) [START_REF] Rupnick | Adipose tissue mass can be regulated through the vasculature[END_REF]. These agents are also able to cause weight reduction in aged, relatively weight-stable ob/ob mice, suggesting that adipose tissue blood vessels are relatively immature and susceptible to inhibitors even when no longer growing. Another study, using life adipose tissue imaging techniques, demonstrated that angiogenesis in obesity requires a close interplay between differentiating adipocytes, stromal cells and blood cells [START_REF] Nishimura | Adipogenesis in obesity requires close interplay between differentiating adipocytes, stromal cells and blood vessels[END_REF]. The use of an anti-VEGF antibody indeed inhibited not only angiogenesis, but also the formation of adipo/angiogenic cell clusters, indicating that coupling of adipogenesis and angiogenesis is essential for differentiation of adipocytes in obesity, and confirming that VEGF is a key mediator [START_REF] Nishimura | Adipogenesis in obesity requires close interplay between differentiating adipocytes, stromal cells and blood vessels[END_REF]. The implication of VEGF in adipose tissue angiogenesis was also evidenced for human tissue, using anti-VEGF antibodies [START_REF] Ledoux | Angiogenesis associated with visceral and subcutaneous adipose tissue in severe human obesity[END_REF]. A recent study showed that blockade of VEGFR-2 but not VEGFR-1, using monoclonal antibodies, can limit diet-induced fat tissue expansion in mice. The results also indicated that angiogenesis from local preexisting vasculature, and not the contribution of bone marrow-derived cells, primarily sustains new vessel formation in fat tissue during diet-induced obesity [START_REF] Tam | Blockade of VEGFR2 and not VEGFR1 can limit diet-induced fat tissue expansion: role of local versus bone marrow-derived endothelial cells[END_REF]. Another strategy to reduce fat mass via its vasculature may be to target prohibitin, a multifunctional membrane protein selectively expressed in adipose tissue endothelial cells [START_REF] Kolonin | Reversal of obesity by targeted ablation of adipose tissue[END_REF]. Ablation of fat tissue can be achieved with a synthetic peptide, that binds to prohibitin and induces apoptosis in adipose tissue blood vessels. Dietary curcumin, the major polyphenol in turmeric spice, may also have a potential benefit in preventing obesity. Supplementing the high-fat diet of mice with curcumin reduced body weight gain, adiposity, and microvessel density in adipose tissue, which coincided with reduced expression of VEGF and VEGFR-2. Curcumin also had a beneficial effect on lipid metabolism [START_REF] Ejaz | Curcumin inhibits adipogenesis in 3T3-L1 adipocytes and angiogenesis and obesity in C57/BL mice[END_REF].

Adipose tissue as a source for therapeutic angiogenesis

Therapeutic angiogenesis using autologous stem/progenitor cells represents a novel strategy for severe ischemic diseases. A number of recent studies, both in animals and humans, demonstrated that implantation of autologous bone marrow mononuclear cells A c c e p t e d M a n u s c r i p t into ischemic skeletal muscles successfully augmented angiogenesis and collateral vessel formation [START_REF] Murohara | Autologous adipose tissue as a new source of progenitor cells for therapeutic angiogenesis[END_REF]. Since bone marrow aspiration is relatively invasive for patients with severe ischemic diseases, less invasive techniques for isolating cells for angiogenic therapy are warranted. Autologous subcutaneous adipose tissue has been proposed as an interesting cell source for therapeutic angiogenesis, since it can be harvested by minimal invasive technology. The stromal vascular fraction of adipose tissue contains multipotent mesenchymal stem/progenitor cells, called adipose-derived stem/progenitor cells (ASCs). These cells can differentiate into various lineages including fibroblasts, adipocytes, pericytes, osteoblasts, chondrocytes, and myocytes, and have the ability to regenerate damaged tissues [START_REF] Zuk | Multilineage cells from human adipose tissue: implications for cell-based therapies[END_REF]. ASCs can also release multiple angiogenesis-related growth factors including VEGF, HGF, and chemokine stromal cellderived factor-1 (SDF-1). The combination of these biological properties (i.e. progenitor cell supply and growth factor release) suggests that autologous subcutaneous adipose tissue may be a next generation candidate as cell source for therapeutic angiogenesis. Murine, as well as human adipose tissue-derived stromal cells, augmented new vessel formation when transplanted into the ischemic hind limb in mice, confirming the potential therapeutic value [START_REF] Rehman | Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells[END_REF][START_REF] Sumi | Transplantation of adipose stromal cells, but not mature adipocytes, augments ischemia-induced angiogenesis[END_REF].

Future lines of investigation

Modulation of angiogenesis appears to have the potential to impair development of obesity. In particular, the VEGF/VEGFR signaling system may be an attractive target. Recent studies have suggested that the expression pattern of pro-and anti-angiogenic components in adipose tissue is fat depot dependent. A better understanding of the regulation of their expression will thus be instrumental in the development of specific targeting approaches. Little is known either on the interactions between these angiogenic components in adipose tissues and on the interplay with adipokines, such as leptin, resistin and adiponectin. The interactions between adipocytes and endothelial cells also remain to be elucidated in more detail. The contribution of inflammatory cell derived cytokines, such as TNF- and several interleukins (e.g. IL-6 and IL-8), to adipogenesis and angiogenesis in adipose tissues also needs further study. Since more and more antiangiogenic agents are characterized in the cancer field, there is a real option to evaluate such components in obesity models in vivo. However, since development of adipose tissue is a complex and multifactorial process, it is unlikely that a single angiogenesis inhibitor will allow reduction of obesity without associated side effects. It should indeed be kept in mind that the angiogenic components involved in adipogenesis and adipose tissue development also are critical to many other biological processes.