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.

PREECLAMPSIA IS A MATERNAL SYNDROME CHARACTERIZED BY THE NEW ONSET OF HYPERTENSION AND PROTEINURIA AFTER 20 WEEKS OF GESTATION ASSOCIATED WITH MULTISYSTEMIC COMPLICATIONS, INCLUDING BRAIN ALTERATIONS. INDEED, BRAIN COMPLICATIONS ASSOCIATED WITH PREECLAMPSIA ARE THE LEADING DIRECT CAUSES OF FETAL AND MATERNAL MORBIDITY AND MORTALITY, ESPECIALLY IN LOW- AND MIDDLE-INCOME COUNTRIES. IN ADDITION TO THE WELL-RECOGNIZED LONG-TERM ADVERSE CARDIOVASCULAR EFFECTS OF PREECLAMPSIA, WOMEN WHO HAVE HAD PREECLAMPSIA HAVE HIGHER RISK OF STROKE, DEMENTIA, INTRACEREBRAL WHITE MATTER LESIONS, EPILEPSY, AND PERHAPS ALSO COGNITIVE DECLINE POSTPARTUM. FURTHERMORE, INCREASING EVIDENCE HAS ALSO ASSOCIATED PREECLAMPSIA WITH SIMILAR COGNITIVE AND CEREBRAL DISORDERS IN THE OFFSPRING. HOWEVER, THE MECHANISTIC LINKS BETWEEN THESE ASSOCIATIONS REMAIN UNRESOLVED. THIS ARTICLE SUMMARIZES THE CURRENT KNOWLEDGE ABOUT THE CEREBROVASCULAR COMPLICATIONS ELICITED BY PREECLAMPSIA AND THE POTENTIAL PATHOPHYSIOLOGICAL MECHANISMS INVOLVED, EMPHASIZING THE IMPAIRED BRAIN VASCULAR FUNCTION IN THE MOTHER AND THEIR OFFSPRING.

TO INVESTIGATE THE FUNCTIONALITY OF A2B ADENOSINE RECEPTOR (A2BAR) AND THE NITRIC OXIDE (NO) AND VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) SIGNALING PATHWAY IN THE ENDOTHELIAL CELL PROLIFERATION/MIGRATION DURING PREECLAMPSIA, WE USED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVECS) ISOLATED FROM NORMAL PREGNANCIES (N = 15) OR PREGNANCIES WITH PREECLAMPSIA (N = 15). EXPERIMENTS WERE PERFORMED IN PRESENCE OR ABSENCE OF THE NONSELECTIVE ADENOSINE RECEPTOR AGONIST NECA, THE A2BAR SELECTIVE ANTAGONIST MRS-1754, AND THE NITRIC OXIDE SYNTHASE (NOS) INHIBITOR L-NAME. RESULTS INDICATED THAT CELLS FROM PREECLAMPSIA EXHIBITED A SIGNIFICANT HIGHER PROTEIN LEVEL OF A2BAR AND LOGEC50 FOR NECA-MEDIATED PROLIFERATION THAN NORMOTENSIVE PREGNANCIES. THE STIMULATORY EFFECT OF NECA (10??M, 24?H) ON CELL PROLIFERATION WAS PREVENTED BY MRS-1754 (5?NM) COINCUBATION ONLY IN CELLS FROM NORMOTENSIVE PREGNANCIES. NEVERTHELESS, L-NAME (100??M, 24?H) REDUCED THE NECA-INDUCED CELL PROLIFERATION/MIGRATION IN HUVEC FROM NORMAL PREGNANCY; HOWEVER IN PREECLAMPSIA ONLY NECA-INDUCED CELL PROLIFERATION WAS REDUCED BY L-NAME. MOREOVER, NECA INCREASED PROTEIN NITRATION AND ABUNDANCE OF VEGF IN CELLS FROM NORMAL PREGNANCY AND EFFECT PREVENTED BY MRS-1754 COINCUBATION. NEVERTHELESS, IN PREECLAMPSIA NECA DID NOT AFFECT THE PROTEIN LEVEL OF VEGF. IN CONCLUSION HUVECS FROM PREECLAMPSIA EXHIBIT ELEVATED PROTEIN LEVEL OF A2BAR AND IMPAIRMENT OF A2BAR-MEDIATED NO/VEGF SIGNALING PATHWAY.

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.

CHILDREN FROM PREGNANCIES AFFECTED BY PREECLAMPSIA HAVE AN INCREASED RISK OF COGNITIVE AND BEHAVIORAL ALTERATIONS VIA UNKNOWN PATHOPHYSIOLOGY. WE TESTED THE HYPOTHESIS THAT PREECLAMPSIA GENERATED REDUCED BRAIN CORTEX ANGIOGENESIS IN THE OFFSPRING.METHODS:THE PREECLAMPSIA-LIKE SYNDROME (PELS) MOUSE MODEL WAS GENERATED BY ADMINISTERING THE NITRIC OXIDE INHIBITOR NG-NITROARGININE METHYL ESTER HYDROCHLORIDE. CONFIRMATORY EXPERIMENTS WERE DONE USING 2 ADDITIONAL PELS MODELS. WHILE IN VITRO ANALYSIS USED MICE AND HUMAN BRAIN ENDOTHELIAL CELLS EXPOSED TO SERUM OF POSTNATAL DAY 5 PUPS OR UMBILICAL PLASMA FROM PREECLAMPTIC PREGNANCIES, RESPECTIVELY.RESULTS:WE REPORT SIGNIFICANT REDUCTION IN THE AREA OCCUPIED BY BLOOD VESSELS IN THE MOTOR AND SOMATOSENSORY BRAIN CORTEX OF OFFSPRING (POSTNATAL DAY 5) FROM PELS COMPARED WITH UNCOMPLICATED CONTROL OFFSPRING. THESE DATA WERE CONFIRMED USING 2 ADDITIONAL PELS MODELS. FURTHERMORE, CIRCULATING LEVELS OF CRITICAL PROANGIOGENIC FACTORS, VEGF (VASCULAR ENDOTHELIAL GROWTH FACTOR), AND PLGF (PLACENTAL GROWTH FACTOR) WERE LOWER IN POSTNATAL DAY 5 PELS. ALSO WE FOUND LOWER VEGF RECEPTOR 2 (KDR [KINASE INSERT DOMAIN-CONTAINING RECEPTOR]) LEVELS IN MICE AND HUMAN ENDOTHELIAL CELLS EXPOSED TO THE SERUM OF POSTNATAL DAY 5 PELS OR FETAL PLASMA OF PREECLAMPTIC PREGNANCIES, RESPECTIVELY. THESE CHANGES WERE ASSOCIATED WITH LOWER IN VITRO ANGIOGENIC CAPACITY, DIMINISHED CELL MIGRATION, LARGER F-ACTIN FILAMENTS, LOWER NUMBER OF FILOPODIA, AND LOWER PROTEIN LEVELS OF F-ACTIN POLYMERIZATION REGULATORS IN BRAIN ENDOTHELIAL CELLS EXPOSED TO SERUM OR FETAL PLASMA OF OFFSPRING FROM PREECLAMPSIA.CONCLUSIONS:OFFSPRING FROM PREECLAMPSIA EXHIBITED DIMINISHED BRAIN CORTEX ANGIOGENESIS, ASSOCIATED WITH LOWER CIRCULATING VEGF/PLGF/KDR PROTEIN LEVELS, IMPAIRED BRAIN ENDOTHELIAL MIGRATION, AND DYSFUNCTIONAL ASSEMBLY OF F-ACTIN FILAMENTS. THESE ALTERATIONS MAY PREDISPOSE TO STRUCTURAL AND FUNCTIONAL ALTERATIONS IN LONG-TERM BRAIN DEVELOPMENT.

OBJECTIVES: ANALYZE WHETHER UMBILICAL CORD PLASMA FROM PREECLAMPTIC PREGNANCIES (U-PE) REDUCES BRAIN ENDOTHELIAL CELL MIGRATION AS AN UNDERLYING MECHANISM OF IMPAIRED BRAIN ANGIOGENESIS. METHODS: HUMAN CEREBRAL MICROVASCULAR ENDOTHELIAL CELLS (HCMEC/D3) WERE INCUBATED (12 H, 1% V/V) WITH UMBILICAL CORD PLASMAS FROM NORMOTENSIVE (N=20, U-NP) OR WOMEN WITH PREECLAMPSIA (N=20, U-PE). CELL VIABILITY (MTT ASSAY), CELL PROLIFERATION (DETECTION OF BROMOURIDINE), AND CELL MIGRATION (WOUND HEALING IN VITRO ASSAY) WERE MEASURED. SYNTHESIS AND RELEASE OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND THE PRESENCE OF ITS RECEPTOR TYPE 2 IN THE INACTIVE (VEGFR2) OR ACTIVE PRO-MIGRATORY PHOSPHORYLATION (Y951-VEGFR2) WERE ANALYZED BY WESTERN BLOT. THE SOLUBLE VEGF RECEPTOR TYPE 1 (OR SFLT-1) WAS MEASURED USING ELISA. ALSO, F-ACTIN POLYMERIZATION/DEPOLYMERIZATION PROTEINS, INCLUDING COFILIN, PHOSPHO-COFILIN, AND ARP2/ARP3 COMPLEX, WERE ANALYZED BY WESTERN BLOT. THE LENGTH AND WIDTH OF F-ACTIN FIBERS (F-ACTIN) WERE EVALUATED USING PHALLOIDIN FLUORESCENCE RESULTS: COMPARED TO PLASMA FROM U-NP, U-PE SIGNIFICANTLY REDUCED CELL PROLIFERATION (P=0.04) AND CELL MIGRATION (P=0.02) WITHOUT AFFECTING CELL VIABILITY. A REDUCTION IN THE VEGFR2 PROTEIN AMOUNT IN BOTH THE INACTIVE AND Y951 PHOSPHORYLATED ISOFORM (PARTICULARLY IN THE CYTOPLASM) WAS FOUND IN CELLS EXPOSED TO U-PE. THIS EFFECT OF U-PE WAS ASSOCIATED WITH REDUCED COFILIN EXPRESSION AND IN THE WIDTH OF F-ACTIN FIBERS, PARTICULARLY IN THE MIDDLE AND TIP SECTIONS OF THE CELLS. IN CONTRAST, NO SIGNIFICANT CHANGES WERE OBSERVED IN THE SYNTHESIS/RELEASE OF VEGF, NEITHER IN THE PLASMA LEVELS NOR THE RELEASE OF SFLT-1. ALSO, NO CHANGES IN PHOSPHO-COFILIN AND ARP2/ARP3 COMPLEX WERE FOUND IN CELLS EXPOSED TO U-PE. CONCLUSION: U-PE LEADS TO REDUCED BRAIN ENDOTHELIAL CELL MIGRATION ASSOCIATED WITH DOWNREGULATION OF THE PRO-MIGRATORY PHOSPHORYLATION OF VEGFR2 AND COFILIN LEVELS THAT MAY LEAD TO HARMED POLYMERIZATION OF F-ACTIN. THESE FINDINGS