Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src-Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day] pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs. (Hypertension. 2012; 59: 1263-1271.). Online Data Supplement

Prenatal retinoic acid improves lung vascularization and VEGF expression in CDH rat

OBJECTIVE: We sought to investigate the effects of antenatal retinoic acid on the pulmonary vasculature and vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) expression in a nitrofen-induced congenital diaphragmatic hernia (CDH) model. STUDY DESIGN: Rat fetuses were exposed to nitrofen at gestational day 9.5 and/or all-trans retinoic acid (ATRA) at gestational days 18.5-20.5. We assessed lung growth, airway, and vascular morphometry. VEGF, VEGFR1, and VEGFR2 expression was analyzed by Western blotting and immunohistochemistry. Continuous data were analyzed by analysis of variance and Kruskal-Wallis test. RESULTS: CDH decreased lung to body weight ratio, increased mean linear intercept and mean transection length/airspace, and decreased mean airspace cord length. ATRA did not affect lung growth or morphometry. CDH increased proportional medial wall thickness of arterioles while ATRA reduced it. ATRA recovered expression of VEGF and receptors, which were reduced in CDH. CONCLUSION: Retinoic acid and VEGF may provide pathways for preventing pulmonary hypertension in CDH.

Interaction between Advanced Glycation End Products Formation and Vascular Responses in Femoral and Coronary Arteries from Exercised Diabetic Rats

Background: The majority of studies have investigated the effect of exercise training (TR) on vascular responses in diabetic animals (DB), but none evaluated nitric oxide (NO) and advanced glycation end products (AGEs) formation associated with oxidant and antioxidant activities in femoral and coronary arteries from trained diabetic rats. Our hypothesis was that 8-week TR would alter AGEs levels in type 1 diabetic rats ameliorating vascular responsiveness. Methodology/Principal Findings: Male Wistar rats were divided into control sedentary (C/SD), sedentary diabetic (SD/DB), and trained diabetic (TR/DB). DB was induced by streptozotocin (i.p.: 60 mg/kg). TR was performed for 60 min per day, 5 days/week, during 8 weeks. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), phenylephrine (PHE) and tromboxane analog (U46619) were obtained. The protein expressions of eNOS, receptor for AGEs (RAGE), Cu/Zn-SOD and Mn-SOD were analyzed. Tissues NO production and reactive oxygen species (ROS) generation were evaluated. Plasma nitrate/nitrite (NOx-), superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and N-epsilon-(carboxymethyl) lysine (CML, AGE biomarker). A rightward shift in the concentration-response curves to ACh was observed in femoral and coronary arteries from SD/DB that was accompanied by an increase in TBARS and CML levels. Decreased in the eNOS expression, tissues NO production and NOx- levels were associated with increased ROS generation. A positive interaction between the beneficial effect of TR on the relaxing responses to ACh and the reduction in TBARS and CML levels were observed without changing in antioxidant activities. The eNOS protein expression, tissues NO production and ROS generation were fully re-established in TR/DB, but plasma NOx- levels were partially restored. Conclusion: Shear stress induced by TR fully restores the eNOS/NO pathway in both preparations from non-treated diabetic rats, however, a massive production of AGEs still affecting relaxing responses possibly involving other endothelium-dependent vasodilator agents, mainly in coronary artery.

Lanthanum carbonate, like sevelamer-HCl, retards the progression of vascular calcification and atherosclerosis in uremic apolipoprotein E-deficient mice

Atherosclerosis and vascular calcification (VC) progression in chronic kidney disease is favored by disturbances of mineral metabolism. We compared the effect of phosphate binder lanthanum (La) carbonate with sevelamer-HCl on atherosclerosis, VC and bone structure and function in mice with chronic renal failure (CRF). Apolipoprotein E-deficient (apoE(-/-)) mice were randomized to one non-CRF and three CRF groups, fed with standard diet (one non-CRF and one CRF) or diet supplemented with either 3% lanthanum carbonate (La3%) or 3% sevelamer-HCl (Sev3%). Both La3% and Sev3% supplemented CRF mice displayed a decrease of serum phosphorus, calcification at both intimal and medial aortic sites and atherosclerosis. This was associated with a reduction of plaque Type I collagen expression by both binders and of positive nitrotyrosine staining in response to sevelamer-HCl only. Increased mineral apposition and bone formation rates in unsupplemented CRF mice were reduced by Sev3% but not by La3%. The beneficial effects of La carbonate and sevelamer-HCl on the progression of VC and atherosclerosis in CRF mice could be mainly due to a decrease in phosphate retention and likewise a reduction of arterial Type I collagen expression. The effect of La carbonate differed from that of sevelamer-HCl in that it did not appear to exert its vascular effects via changes in oxidative stress or bone remodeling in the present model.

Identification of protein expression signatures in gastric carcinomas using clustering analysis

Background and Aim: The identification of gastric carcinomas (GC) has traditionally been based on histomorphology. Recently, DNA microarrays have successfully been used to identify tumors through clustering of the expression profiles. Random forest clustering is widely used for tissue microarrays and other immunohistochemical data, because it handles highly-skewed tumor marker expressions well, and weighs the contribution of each marker according to its relatedness with other tumor markers. In the present study, we e identified biologically- and clinically-meaningful groups of GC by hierarchical clustering analysis of immunohistochemical protein expression. Methods: We selected 28 proteins (p16, p27, p21, cyclin D1, cyclin A, cyclin B1, pRb, p53, c-met, c-erbB-2, vascular endothelial growth factor, transforming growth factor [TGF]-beta I, TGF-beta II, MutS homolog-2, bcl-2, bax, bak, bcl-x, adenomatous polyposis coli, clathrin, E-cadherin, beta-catenin, mucin (MUC) 1, MUC2, MUC5AC, MUC6, matrix metalloproteinase [ MMP]-2, and MMP-9) to be investigated by immunohistochemistry in 482 GC. The analyses of the data were done using a random forest-clustering method. Results: Proteins related to cell cycle, growth factor, cell motility, cell adhesion, apoptosis, and matrix remodeling were highly expressed in GC. We identified protein expressions associated with poor survival in diffuse-type GC. Conclusions: Based on the expression analysis of 28 proteins, we identified two groups of GC that could not be explained by any clinicopathological variables, and a subgroup of long-surviving diffuse-type GC patients with a distinct molecular profile. These results provide not only a new molecular basis for understanding the biological properties of GC, but also better prediction of survival than the classic pathological grouping.

Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

Abstract Background Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings. Methods Male Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70–80% VO2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx-) were measured. Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting. Results High caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl). Neither high caloric diet nor RT modified NOx- levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels. Conclusion The improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation.

VEGF-C expression in oral cancer by neurotransmitter-induced activation of beta-adrenergic receptors

The aim of this study was to investigate the expression of vascular endothelial growth factor type C (VEGF-C) in oral squamous cell carcinoma (OSCC) cell lines through norepinephrine-induced activation of beta-adrenergic receptors. Human OSCC cell lines (SCC-9 and SCC-25) expressing beta-adrenergic receptors were stimulated with different concentrations of norepinephrine (0.1, 1, and 10 μM) and 1 μMof propranolol, and analyzed after 1, 6, and 24 h. VEGF-C gene expression and VEGF-C production in the cell supernatant were evaluated by real-time PCR and by ELISA, respectively. The results showed that beta-adrenergic receptor stimulation by different concentrations of norepinephrine or blocking by propranolol did not markedly alter VEGF-C expression by SCC-9 and SCC-25 cells. VEGF-C protein levels produced by oral malignant cell lines after stimulation with different norepinephrine concentrations or blocking with propranolol was statistically similar (p>0.05) to those of the control group (nonstimulated OSCC cell lines). Our findings suggest that stimulation of beta-adrenergic receptors by means of norepinephrine does not seem to modulate the VEGF-C expression in OSCC cell lines. These findings reinforce the need for further studies in order to understand the responsiveness of oral cancer to beta-adrenergic receptor stimulation or blockage, especially with regard to VEGF-C production.

Effects of the administration of pentoxifylline and prednisolone on the evolution of portal fibrogenesis secondary to biliary obstruction in growing animals: immunohistochemical analysis of the expression of TGF-β and VEGF

OBJECTIVE: During the neonatal and infancy periods, some chronic liver diseases may lead to progressive hepatic fibrosis, which is a condition that can ultimately result in the loss of organ function and severe portal hypertension necessitating hepatic transplantation. In a previous report, pharmacological interventions were demonstrated to modulate hepatic fibrosis induced by bile duct ligation in young rats. The administration of pentoxifylline or prednisolone, or the combination of both, resulted in reduced fibrogenesis in portal spaces. The objectives of the present study were to evaluate the expression of transforming growth factor β and vascular endothelial growth factor after bile duct ligation in young rats and to assess the effect of those same drugs on cytokine expression. METHODS: In this experimental study, 80 young rats (21 or 22 days old) were submitted either to laparotomy and common bile duct ligation or to sham surgery. The animals were allocated into four groups according to surgical procedure, and the following treatments were administered: (1) common bile duct ligation + distilled water, (2) sham surgery + distilled water, (3) common bile duct ligation + pentoxifylline, or (4) common bile duct ligation + prednisolone. After 30 days, a hepatic fragment was collected from each animal for immunohistochemical analysis using monoclonal antibodies against transforming growth factor β and vascular endothelial growth factor. Digital morphometric and statistical analyses were performed. RESULTS: The administration of pentoxifylline reduced the transforming growth factor β-marked area and the amount of transforming growth factor β expressed in liver tissue. This effect was not observed after the administration of prednisolone. There was a significant reduction in vascular endothelial growth factor expression after the administration of either drug compared with the non-treatment group. CONCLUSIONS: The administration of pentoxifylline to cholestatic young rats resulted in the diminished expression of transforming growth factor β and vascular endothelial growth factor in liver tissue. The administration of steroids resulted in the diminished expression of vascular endothelial growth factor only. These pathways may be involved in hepatic fibrogenesis in young rats submitted to bile duct ligation and exposed to pentoxifylline or prednisolone.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D 10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N ¼ 42) or not (small follicle-small CL group; SF-SCL; N ¼ 41) on D 10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 0.33 mm vs. 10.76 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 0.28 pg/mL vs. 1.27 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 0.25 ng/mL vs. 2.62 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid deltaisomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Acclimatization to 4100 m does not change capillary density or mRNA expression of potential angiogenesis regulatory factors in human skeletal muscle

[EN] Increased skeletal muscle capillary density would be a logical adaptive mechanism to chronic hypoxic exposure. However, animal studies have yielded conflicting results, and human studies are sparse. Neoformation of capillaries is dependent on endothelial growth factors such as vascular endothelial growth factor (VEGF), a known target gene for hypoxia inducible factor 1 (HIF-1). We hypothesised that prolonged exposure to high altitude increases muscle capillary density and that this can be explained by an enhanced HIF-1alpha expression inducing an increase in VEGF expression. We measured mRNA levels and capillary density in muscle biopsies from vastus lateralis obtained in sea level residents (SLR; N=8) before and after 2 and 8 weeks of exposure to 4100 m altitude and in Bolivian Aymara high-altitude natives exposed to approximately 4100 m altitude (HAN; N=7). The expression of HIF-1alpha or VEGF mRNA was not changed with prolonged hypoxic exposure in SLR, and both genes were similarly expressed in SLR and HAN. In SLR, whole body mass, mean muscle fibre area and capillary to muscle fibre ratio remained unchanged during acclimatization. The capillary to fibre ratio was lower in HAN than in SLR (2.4+/-0.1 vs 3.6+/-0.2; P<0.05). In conclusion, human muscle VEGF mRNA expression and capillary density are not significantly increased by 8 weeks of exposure to high altitude and are not increased in Aymara high-altitude natives compared with sea level residents.

Angiogenesis and angioregression gene expression analyses in swine corpus luteum

The corpus luteum (CL) lifespan is characterized by a rapid growth, differentiation and controlled regression of the luteal tissue, accompanied by an intense angiogenesis and angioregression. Indeed, the CL is one of the most highly vascularised tissue in the body with a proliferation rate of the endothelial cells 4- to 20-fold more intense than in some of the most malignant human tumours. This angiogenic process should be rigorously controlled to allow the repeated opportunities of fertilization. After a first period of rapid growth, the tissue becomes stably organized and prepares itself to switch to the phenotype required for its next apoptotic regression. In pregnant swine, the lifespan of the CLs must be extended to support embryonic and foetal development and vascularisation is necessary for the maintenance of luteal function. Among the molecules involved in the angiogenesis, Vascular Endothelial Growth Factor (VEGF) is the main regulator, promoting endothelial cells proliferation, differentiation and survival as well as vascular permeability and vessel lumen formation. During vascular invasion and apoptosis process, the remodelling of the extracellular matrix is essential for the correct evolution of the CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). Another important factor that plays a role in the processes of angiogenesis and angioregression during the CL formation and luteolysis is the isopeptide Endothelin-1 (ET-1), which is well-known to be a potent vasoconstrictor and mitogen for endothelial cells. The goal of the present thesis was to study the role and regulation of vascularisation in an adult vascular bed. For this purpose, using a precisely controlled in vivo model of swine CL development and regression, we determined the levels of expression of the members of VEGF system (VEGF total and specific isoforms; VEGF receptor-1, VEGFR-1; VEGF receptor-2, VEGFR-2) and ET- 1 system (ET-1; endothelin converting enzyme-1, ECE-1; endothelin receptor type A, ET-A) as well as the activity of the Ca++/Mg++-dependent endonucleases and gelatinases (MMP-2 and MMP-9). Three experiments were conducted to reach such objectives in CLs isolated from ovaries of cyclic, pregnant or fasted gilts. In the Experiment I, we evaluated the influence of acute fasting on VEGF production and VEGF, VEGFR-2, ET-1, ECE-1 and ET-A mRNA expressions in CLs collected on day 6 after ovulation (midluteal phase). The results indicated a down-regulation of VEGF, VEGFR-2, ET-1 and ECE-1 mRNA expression, although no change was observed for VEGF protein. Furthermore, we observed that fasting stimulated steroidogenesis by luteal cells. On the basis of the main effects of VEGF (stimulation of vessel growth and endothelial permeability) and ET-1 (stimulation of endothelial cell proliferation and vasoconstriction, as well as VEGF stimulation), we concluded that feed restriction possibly inhibited luteal vessel development. This could be, at least in part, compensated by a decrease of vasal tone due to a diminution of ET-1, thus ensuring an adequate blood flow and the production of steroids by the luteal cells. In the Experiment II, we investigated the relationship between VEGF, gelatinases and Ca++/Mg++-dependent endonucleases activities with the functional CL stage throughout the oestrous cycle and at pregnancy. The results demonstrated differential patterns of expression of those molecules in correspondence to the different phases of the oestrous cycle. Immediately after ovulation, VEGF mRNA/protein levels and MMP-9 activity are maximal. On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed. These results suggested that during the very early luteal phase, high MMPs activities coupled with high VEGF levels drive the tissue to an angiogenic phenotype, allowing CL growth under LH (Luteinising Hormone) stimulus, while during the late luteal phase, low VEGF and elevate MMPs levels may play a role in the apoptotic tissue and extracellular matrix remodelling during structural luteolysis. In the Experiment III, we described the expression patterns of all distinct VEGF isoforms throughout the oestrous cycle. Furthermore, the mRNA expression and protein levels of both VEGF receptors were also evaluated. Four novel VEGF isoforms (VEGF144, VEGF147, VEGF182, and VEGF164b) were found for the first time in swine and the seven identified isoforms presented four different patterns of expression. All isoforms showed their highest mRNA levels in newly formed CLs (day 1), followed by a decrease during mid-late luteal phase (days 10–17), except for VEGF182, VEGF188 and VEGF144 that showed a differential regulation during late luteal phase (day 14) or at luteolysis (day 17). VEGF protein levels paralleled the most expressed and secreted VEGF120 and VEGF164 isoforms. The VEGF receptors mRNAs showed a different pattern of expression in relation to their ligands, increasing between day 1 and 3 and gradually decreasing during the mid-late luteal phase. The differential regulation of some VEGF isoforms principally during the late luteal phase and luteolysis suggested a specific role of VEGF during tissue remodelling process that occurs either for CL maintenance in case of pregnancy or for noncapillary vessel development essential for tissue removal during structural luteolysis. In summary, our findings allow us to determine relationships among factors involved in the angiogenesis and angioregression mechanisms that take place during the formation and regression of the CL. Thus, CL provides a very interesting model for studying such factors in different fields of the basic research.

Espressione del vascular endothelium growth factor (VEGF) nella micosi fungoide

La micosi fungoide (MF) è un linfoma a cellule T primitivo della cute usualmente indolente negli stadi iniziali, ma con prognosi decisamente peggiore nelle fasi avanzate, ove attualmente non sono presenti strategie terapeutiche tali da indurre remissioni durature. Recenti osservazioni indicano che alti livelli di espressione del vascular endothelial growth factor (VEGF) nelle cellule della MF sembrano correlare con una prognosi peggiore. Nel presente studio, sono state vagliate le eventuali differenze di espressione di VEGF nella MF e nei linfociti T normali. In primo luogo sono stati raffrontati 63 casi di MF con 20 campioni corrispondenti alle diverse sottopopolazioni di linfociti T normali, per stabilire quale fra questi esprimesse maggiori livelli di VEGF. Tale esperimento ha dimostrato che il gene è notevolmente più espresso nella MF. Si è provveduto a stabilire se tale dato sia da correlarsi ad un fenomeno patologico o fisiologico. Quindi sono state eseguite indagini di gene expression profiling (GEP) allo scopo di vagliare i livelli di VEGF nella popolazione linfocitaria T normale (CD4+, CD8+, HLA-DR+ e HLA-DR-): da ciò è risultato che i linfociti T attivati esprimono maggiormente VEGF e che il loro GEP è globalmente paragonabile a quello della MF. Pertanto, i linfociti T attivati sono stati considerati la controparte normale delle cellule della MF. Successivamente si è confrontata l’espressione quantitativa di VEGF nei linfociti T attivati e nelle cellule della MF, evidenziando come questa sia maggiore nella popolazione neoplastica indipendentemente dallo stadio della malattia. Le indagini immunoistochimiche condotte su 18 casi di MF, hanno confermato quanto evidenziato attraverso il GEP. Concludendo, la ricerca ha dimostrato per la prima volta l’espressione di VEGF negli elementi della MF. Ciò porta a supporre che la de-regolazione genica della via di VEGF sia correlata nella patogenesi della MF e che tale molecola possa considerarsi un potenziale bersaglio terapeutico.

Zelluläre Kontrolle adulter Neurogenese - Expression und Funktion des VEGF-Rezeptor-1 (Flt-1) im adulten Säugerhirn

Neurale Stammzellen sind im adulten Säugerhirn in der Subventrikulären Zone (SVZ) der Lateralventrikel und dem Hippokampus lokalisiert. In der SVZ entstandene neurale Zellen migrieren entlang eines von Astrozyten umgebenen Pfades, dem Rostralmigratorischen Strom (RMS), zum Olfaktorischen Bulbus (OB), wo sie zu olfaktorischen Interneuronen differenzieren. Vaskuläre Wachstumsfaktoren, wie VEGF-A beeinflussen die adulte Neurogenese. Die vorliegende Arbeit beschreibt erstmalig detailliert die spezifische Expression des VEGF-Rezeptor-1 (VEGFR-1) in den Regionen olfaktorischer und hippokampaler Neurogenese des adulten ZNS. Die Ergebnisse zeigen, dass VEGFR-1 im adulten Hirn hauptsächlich in GFAP-positiven Zellen in der SVZ, dem RMS, dem OB, dem Corpus callosum und dem Hippokampus exprimiert ist. In vivo-Analysen transgener Mäuse (Flt-1TK-/-), denen die Signaltransduktionsdomäne des VEGFR-1 fehlt, demonstrieren hier erstmals eine Rolle des VEGFR-1 in adulter Neurogenese. Flt-1TK-/- weisen eine erhöhte Proliferation neuronaler Vorläuferzellen der SVZ auf. Im RMS ist jedoch 6 Tage nach BrdU-Administration die Anzahl markierter Zellen im Vergleich zum Wildtyp (wt) um 47,97% reduziert, ohne dass es zu einer Akkumulation in der SVZ kommt. Zusammen mit der in Kulturversuchen stark erhöhten Migrationsgeschwindigkeit von Neuroblasten der Flt-1TK-/- und einer verminderten Abwanderung von Zellen aus dem RMS ins Corpus callosum der Flt-1Tk-/-, weist dies auf eine gesteigerte Migration zum OB hin. Tatsächlich war der OB der Flt-1TK-/-, vor allem die Plexiform- und Periglomerulärzellschicht (PGL), signifikant vergrößert. Im OB der transgenen Tiere migrieren zudem signifikant mehr BrdU-markierte Zellen in die PGL. Dort differenzieren signifikant mehr Neurone als im wt. Subtypisierungen zeigen, zudem eine erhöhte Differenzierung in dopaminerge Interneurone in der PGL der Flt-1TK-/-. Im Gehirn Flt-1TK-/- war die Konzentration von VEGF-A erhöht. Intrazerebroventrikuläre Infusion von VEGF-A in wt-Tiere erbrachte den eindeutigen Nachweis, dass die Erhöhung der VEGF-A-Konzentration im Gehirn der Flt-1TK-/- ursächlich für die in diesen Tieren beobachtete Reduktion der BrdU-positiven Zellen im RMS ist. Dies ist gleichzeitig der erste Nachweis einer Wirkung von VEGF-A auf Neuroblasten im RMS in vivo unter physiologischen Bedingungen. Die erhöhte VEGF-A-Konzentration könnte auch den anderen hier dargelegten Effekten zugrunde liegen. VEGFR-1 ist somit ein regulatorischer Faktor für die adulte olfaktorische Neurogenese und spielt eine potentielle Rolle in der Differenzierung dopaminerger Interneurone.