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.

OBJECTIVES: INVESTIGATE WHETHER SMALL EXTRACELLULAR VESICLES (PDSEVS) EXTRACTED FROM PLACENTAS CULTURED IN HYPOXIA DISRUPT THE BLOOD-BRAIN BARRIER (BBB) IN SELECTED AREAS OF THE BRAIN. METHODS: PDSEVSWERE EXTRACTED FROM PLACENTAL EXPLANTS OF NORMAL PREGNANCY CULTURED IN NORMOXIA (21% O2) OR HYPOXIA (1% O2, 18 H). PDSEVS (100 MG OF PROTEIN) FROM NORMOXIA (N=6) OR HYPOXIA (N=6) WERE INJECTED IN NON-PREGNANT FEMALE C57BL/7 MICE. INJECTED MICE WERE EVALUATED USING THE RAPID MURINE COMA AND BEHAVIOR SCALE (RMSBS, 0-24 H), WHILE SYSTEMIC BLOOD PRESSURE WAS MEASURED BEFORE AND AFTER TREATMENT. MICE WERE INJECTED WITH EVAN S BLUE AND THEN EUTHANIZED TO EXTRACT THE BRAIN. MICE BRAINS WERE WEIGHED AND SECTIONED (100 MM SLICES) TO ANALYZE EVAN S BLUE EXTRAVASATION. ALSO, HUMAN CEREBRAL MICROVASCULAR ENDOTHELIAL CELLS (HCMEC/D3) WERE EXPOSED TO PDSEVS. BBB PARAMETERS INCLUDED ELECTRICAL RESISTANCE (TEER) AND CELL PERMEABILITY TO A HIGH-MOLECULAR-WEIGHT FLUORESCENT DYE. ALSO, THE AMOUNT OF CLAUDIN-5 IN THE CELL MEMBRANE WAS ANALYZED BY WESTERN BLOT. UPTAKE OF PDSEVS WAS ANALYZED USING PKH67. RESULTS: MICE TREATED WITH HYPOXIC PDSEVS EXHIBITED NEUROLOGICAL DEFICITS (FROM 3 TO 24 H), AS INDICATED WITH A SIGNIFICANT REDUCTION IN THE SCORE FOR SPINAL REFLEXES AND MOTOR SKILLS, WHICH WAS ASSOCIATED WITH MORE SIGNIFICANT EXTRAVASATION OF EVAN S BLUE, MAINLY IN THE ANTERIOR PORTION OF THE BRAIN. THERE WERE NO SIGNIFICANT CHANGES IN SYSTOLIC/DIASTOLIC BLOOD PRESSURE IN ANY OF THE TREATED GROUPS. HYPOXIC PDSEVS GENERATED A MORE SIGNIFICANT REDUCTION IN THE TEER VALUES (P

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