WE AIM TO INVESTIGATE WHETHER A2A/NITRIC OXIDE-MEDIATED REGULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) EXPRESSION IS IMPAIRED IN FETO-PLACENTAL ENDOTHELIAL CELLS FROM LATE-ONSET PRE-ECLAMPSIA. CULTURES OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVECS) AND HUMAN PLACENTAL MICROVASCULAR ENDOTHELIAL CELLS (HPMECS) FROM NORMAL AND PRE-ECLAMPTIC PREGNANCIES WERE USED. ASSAYS BY USING SMALL INTERFERENCE RNA (SIRNA) FOR A2A WERE PERFORMED, AND TRANSFECTED CELLS WERE USED FOR ESTIMATION OF MESSENGER RNA (MRNA) LEVELS OF VEGF, AS WELL AS FOR CELL PROLIFERATION AND ANGIOGENESIS IN VITRO. CGS-21680 (A2A AGONIST, 24 H) INCREASES HUVEC AND HPMEC PROLIFERATION IN A DOSE RESPONSE MANNER. FURTHERMORE, SIMILAR TO CGS-21680, THE NITRIC OXIDE DONOR, S-NITROSO-N-ACETYL-PENICILLAMINE OXIDE (SNAP), INCREASED CELL PROLIFERATION IN A DOSE RESPONSE MANNER (LOGEC50 10-9.2 M). IN HPMEC, CGS-21680 INCREASED VEGF PROTEIN LEVELS IN BOTH NORMAL (?1.5-FOLD) AND PRE-ECLAMPTIC PREGNANCIES (?1.2-FOLD), AN EFFECT BLOCKED BY THE A2A ANTAGONIST, ZM-241385 (10-5 M) AND THE INHIBITOR OF NO SYNTHASE, N ?-NITRO-L-ARGININE METHYL ESTER HYDROCHLORIDE (L-NAME). SUBSEQUENTLY, SNAP PARTIALLY RECOVERED CELL PROLIFERATION AND IN VITRO ANGIOGENESIS CAPACITY OF CELLS FROM NORMAL PREGNANCIES EXPOSED TO SIRNA FOR A2A. CGS-21680 ALSO INCREASED (?1.5-FOLD) THE LEVEL OF VEGF MRNA IN HUVEC FROM NORMAL PREGNANCIES, BUT NOT IN PRE-ECLAMPSIA. ADDITIONALLY, TRANSFECTION WITH SIRNA FOR A2A DECREASE (?30 %) THE LEVEL OF MRNA FOR VEGF IN NORMAL PREGNANCY COMPARED TO UNTRANSFECTED CELLS, AN EFFECT PARTIALLY REVERSED BY CO-INCUBATION WITH SNAP. THE A2A-NO-VEGF PATHWAY IS PRESENT IN ENDOTHELIUM FROM MICROCIRCULATION AND MACROCIRCULATION IN BOTH NORMAL AND PRE-ECLAMPTIC PREGNANCIES. HOWEVER, NO SIGNALING PATHWAY SEEMS TO BE IMPAIRED IN HUVEC FROM PRE-ECLAMPSIA.

ESTROGENIC STEROIDS AND ADENOSINE A2A RECEPTORS PROMOTE THE WOUND HEALING AND ANGIOGENESIS PROCESSES. HOWEVER, SO FAR, IT IS UNCLEAR WHETHER ESTROGEN MAY REGULATE THE EXPRESSION AND PRO-ANGIOGENIC ACTIVITY OF A2A RECEPTORS. USING IN VIVO ANALYSES, WE SHOWED THAT FEMALE WILD TYPE (WT) MICE HAVE A MORE RAPID WOUND HEALING PROCESS THAN FEMALE OR MALE A2A-DEFICIENT MICE (A2AKO) MICE. WE ALSO FOUND THAT PULMONARY ENDOTHELIAL CELLS (MPEC) ISOLATED FROM FEMALE WT MICE SHOWED HIGHER EXPRESSION OF A2A RECEPTOR THAN MPEC FROM MALE WT MICE. MPEC FROM FEMALE WT MICE WERE MORE SENSITIVE TO A2A-MEDIATED PRO-ANGIOGENIC RESPONSE, SUGGESTING AN ER AND A2A CROSSTALK, WHICH WAS CONFIRMED USING CELLS ISOLATED FROM A2AKO. IN THOSE FEMALE CELLS, 17?-ESTRADIOL POTENTIATED A2A-MEDIATED CELL PROLIFERATION, AN EFFECT THAT WAS INHIBITED BY SELECTIVE ANTAGONISTS OF ESTROGEN RECEPTORS (ER), ER?, AND ER?. THEREFORE, ESTROGEN REGULATES THE EXPRESSION AND/OR PRO-ANGIOGENIC ACTIVITY OF A2A ADENOSINE RECEPTORS, LIKELY INVOLVING ACTIVATION OF ER? AND ER? RECEPTORS. SEXUAL DIMORPHISM IN WOUND HEALING OBSERVED IN THE A2AKO MICE PROCESS REINFORCES THE FUNCTIONAL CROSSTALK BETWEEN ER AND A2A RECEPTORS.

OBJECTIVE: WE AIMED TO INVESTIGATE WHETHER FETO-PLACENTAL TISSUE FROM GESTATIONAL DIABETES MELLITUS (GDM) EXHIBITS AN ELEVATED PRO-ANGIOGENIC PHENOTYPE COMPARED TO NORMAL PREGNANCY. METHODS: WE OBTAINED PLACENTAL SAMPLES FROM NORMAL PREGNANT WOMEN (N=12) AND GDM PATIENTS (N=14), WHICH WERE HOMOGENIZED TO OBTAIN MRNA AND PROTEIN EXTRACTIONS. SEMI-QUANTITATIVE PCR WAS PERFORMED TO ESTIMATE MRNA LEVELS OF VEGF, VEGFR TYPE 1 (VEGFR1) AND VEGFR2. ALSO, PROTEIN CONCENTRATIONS OF CD31, CD34, KI67, VEGF, VEGFR1, VEGFR2 AND Y951-PHOSPHORYLATED VEGFR2 WERE ESTIMATED BY WESTERN BLOT. Y1175 PHOSPHORYLATED VEGFR2 WAS MEASURED BY ELISA. ALSO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) WERE ISOLATED FROM NORMAL (N=10) AND GDM (N=10) WOMAN. CELL PROLIFERATION WAS ANALYZED BY COLORIMETRIC MTS-ASSAY (PROMEGA, USA), WHEREAS MIGRATION WAS ANALYZED BY CELL-SCRATCH ASSAY AFTER 24 HOURS OF CELL INCUBATION WITHOUT ANY TREATMENT.

PLACENTAS FROM GESTATIONAL DIABETES MELLITUS (GDM) ARE OFTEN HYPERVASCULARIZED; HOWEVER, PARTICIPATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND ITS RECEPTORS IN THIS PLACENTAL ADAPTATION IS UNCLEAR. WE AIMED TO TEST WHETHER CHANGES IN PHOSPHORYLATION OF TYROSINE 951 OR TYROSINE 1175 (PY951 OR PY1175) OF THE VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 (KDR) ARE ASSOCIATED WITH THE PROANGIOGENIC STATE OBSERVED IN PLACENTAS FROM GDM. WE OBTAINED PLACENTAL SAMPLES FROM WOMEN WITH NORMAL PREGNANCIES (N = 24) OR GDM (N = 18). WE MEASURED THE RELATIVE EXPRESSION OF MARKERS FOR ENDOTHELIAL CELL NUMBER (CD31, CD34), VEGF, VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 1 (FLT-1), KDR, PY951 AND PY1175 OF KDR IN PLACENTAL HOMOGENATE. IMMUNOHISTOCHEMISTRY OF PLACENTAL BLOOD VESSELS WERE PERFORMED USING CD34. PROLIFERATION AND MIGRATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) OBTAINED FROM NORMAL PREGNANCY AND GDM WERE DETERMINED IN ABSENCE OR PRESENCE OF CONDITIONED MEDIUM (CM) HARVESTED FROM GDM OR NORMOGLYCEMIC HUVEC CULTURES. GDM WAS ASSOCIATED WITH MORE CD31 AND CD34 PROTEIN COMPARED TO NORMAL PREGNANCY. HIGH NUMBER, BUT REDUCED AREA OF PLACENTAL BLOOD VESSELS WAS FOUND IN GDM. REDUCED FLT-1 LEVELS (MRNA AND PROTEIN) ARE ASSOCIATED WITH REDUCED KDR MRNA, BUT HIGHER KDR PROTEIN LEVELS IN PLACENTAS FROM GDM. NO SIGNIFICANT CHANGES IN Y951-OR Y1175-PHOSPHORYLATION OF KDR IN PLACENTAS FROM GDM WERE FOUND. GDM DID NOT ALTER PROLIFERATION OF HUVECS, BUT ENHANCED MIGRATION. CONDITIONED MEDIUM HARVESTED FROM GDM HUVEC CULTURES ENHANCED KDR PROTEIN AMOUNT, TUBE FORMATION CAPACITY AND CELL MIGRATION IN HUVEC ISOLATED FROM NORMOGLYCEMIC PREGNANCIES. THE DATA INDICATE THAT GDM IS ASSOCIATED WITH REDUCED EXPRESSION OF FLT-1 BUT HIGH PRO-MIGRATORY ACTIVATION OF KDR REFLECTING A PROANGIOGENIC STATE IN GDM.

INDIRECT EVIDENCES SUGGEST REDUCED ANGIOGENESIS IN OFFSPRING BORN TO PREECLAMPSIA. ADENOSINE, VIA NITRIC OXIDE (NO) AND VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) MAY REGULATE ANGIOGENESIS. OBJECTIVES: TO INVESTIGATE WHETHER A2A ADENOSINE RECEPTOR (A2AAR)/NO/VEGF EXPRESSION IS IMPAIRED IN OFFSPRING FROM PRE-ECLAMPSIA. METHODS: IN HUMAN, CULTURES OF UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) AND PLACENTAL MICROVASCULAR ENDOTHELIAL CELLS (HPMEC) FROM NORMAL PREGNANCIES AND WOMEN WITH PREECLAMPSIA WERE USED FOR ESTIMATION OF MRNA LEVELS OF VEGF, CELL PROLIFERATION AND ANGIOGENESIS IN VITRO IN PRESENCE OR ABSENCE OF SIRNA FOR A2AAR. ALSO, PREECLAMPSIA-LIKE SYNDROME WAS GENERATED IN PREGNANT MICE (C56BJ) EXPOSED TO THE NO SYNTHASE INHIBITOR, NG-NITRO-L-ARGININE METHYL ESTER (L-NAME, 50 MG/KG). IN VIVO BLOOD PERFUSION USING LASER DOPPLER WAS ANALYSED AT DAY 7 POSTNATAL IN OFFSPRING (F1), EXPOSED OR NOT TO PREECLAMPSIA-LIKE SYNDROME.

THE UNDERLYING MOLECULAR MECHANISMS INVOLVE IN THE REGULATION OF THE ANGIOGENIC PROCESS BY INSULIN ARE NOT WELL UNDERSTOOD. IN THIS REVIEW ARTICLE, WE AIM TO DESCRIBE THE ROLE OF INSULIN AND INSULIN RECEPTOR ACTIVATION ON THE CONTROL OF ANGIOGENESIS AND HOW THESE MECHANISMS CAN BE DEREGULATED IN HUMAN DISEASES. FUNCTIONAL EXPRESSION OF INSULIN RECEPTORS AND THEIR SIGNALING PATHWAYS HAS BEEN DESCRIBED ON ENDOTHELIAL CELLS AND PERICYTES, BOTH OF THE MAIN CELLS INVOLVED IN VESSEL FORMATION AND MATURATION. CONSEQUENTLY, INSULIN HAS BEEN SHOWN TO REGULATE ENDOTHELIAL CELL MIGRATION, PROLIFERATION, AND IN VITRO TUBULAR STRUCTURE FORMATION THROUGH BINDING TO ITS RECEPTORS AND ACTIVATION OF INTRACELLULAR PHOSPHORYLATION CASCADES. FURTHERMORE, INSULIN-MEDIATED PRO-ANGIOGENIC STATE IS POTENTIATED BY GENERATION OF VASCULAR GROWTH FACTORS, SUCH AS THE VASCULAR ENDOTHELIAL GROWTH FACTOR, PRODUCED BY ENDOTHELIAL CELLS. ADDITIONALLY, DISEASES SUCH AS INSULIN RESISTANCE, OBESITY, DIABETES, AND CANCER MAY BE ASSOCIATED WITH THE DEREGULATION OF INSULIN-MEDIATED ANGIOGENESIS. DESPITE THIS KNOWLEDGE, THE UNDERLYING MOLECULAR MECHANISMS NEED TO BE ELUCIDATED IN ORDER TO PROVIDE NEW INSIGHTS INTO THE ROLE OF INSULIN ON ANGIOGENESIS.

LIPOINFLAMATION IS THE INFLAMMATION GENERATED IN THE ADIPOSE TISSUE. IT CAN CONTRIBUTE TO THE DEVELOPMENT OF INSULIN RESISTANCE. THE LIPOINFLAMMATION-ASSOCIATED MECHANISMS ARE RELATED TO THE FUNCTION OF ADIPOCYTES AND MACROPHAGES PRESENT IN THE ADIPOSE TISSUE. IN THIS REGARD, THE LEVEL OF NUCLEOSIDE ADENOSINE IS INCREASED IN INDIVIDUALS WITH OBESITY. CAUSES OR CONSEQUENCES OF THIS INCREASE ARE UNKNOWN. ALTHOUGH, ADENOSINE ACTIVATING ITS RECEPTORS (A1, A2A, A2B AND A3) IS ABLE TO DIFFERENTIALLY MODULATE THE FUNCTION OF ADIPOCYTES AND MACROPHAGES, IN ORDER TO AVOID THE REDUCTION OF INSULIN SENSITIVITY AND GENERATE AN ANTI-INFLAMMATORY STATE IN SUBJECT WITH OBESITY. IN THIS REVIEW WE PROPOSE THAT ADENOSINE COULD BE A KEY ELEMENT IN THE DEVELOPMENT OF NEW STRATEGIES FOR LIMIT LIPOINFLAMMATION AND REGULATE METABOLIC HOMEOSTASIS THROUGH MODULATION OF ADIPOCYTE-MACROPHAGE DIALOGUE.