MICROCIRCULATION ANALYSIS OF THE BRAIN CORTEX IS CHALLENGING BECAUSE SURFACE PERFUSION VARIES RAPIDLY IN SMALL SPACE-TIME REGIONS AND IS BONE PROTECTED. THE LASER SPECKLE CONTRAST IMAGING (LSCI) TECHNIQUE ALLOWS ANALYZING IN VIVO BRAIN VASCULAR PERFUSION GENERATING A LARGE AMOUNT OF DATA THAT REQUIRES SOPHISTICATED DATA ANALYTICS, MAKING RESEARCHERS INVEST MUCH EFFORT IN PROCESSING. OUR RESEARCH QUESTION WAS WHETHER THE REDUCED PLACENTAL PERFUSION MODEL (RUPP) OF PREECLAMPSIA (PE) WAS ASSOCIATED WITH IMPAIRED BLOOD PERFUSION IN THE OFFSPRING'S BRAINS. WE AIMED TO DEVELOP A ROBUST NUMERICAL APPROACH THAT MAINLY CONSISTED OF APPLYING A SIGNAL-PROCESSING TOOL FOR CALCULATING OPTIMAL SEGMENTATION AND PIECE-WISE FITS OF THE OFFSPRING'S BRAIN PERFUSION SIGNALS OBTAINED FROM THE LSCI TECHNIQUE. WE COMBINED THIS TOOL WITH THE USUAL STATISTICAL ANALYSIS, IMPLEMENTING BOTH IN MATLAB SOFTWARE. WE PERFORMED BRAIN PERFUSION MEASUREMENTS FROM OFFSPRING (FIVE DAYS POSTNATAL, P5) OF CONTROL PREGNANT DAMS (SHAM, N = 13) AND OF RUPP DAMS (RUPP, N = 7) USING THE PERICAM® PSI-HR SYSTEM AT A BASAL CONDITION AND AFTER THERMAL STIMULI (WARM AND COLD). WE FOUND THAT PUPS OF RUPP MICE EXHIBITED SIGNIFICANT DIFFERENCES IN PERFUSION AND VASCULAR RESPONSE TO THERMAL STIMULI COMPARED TO THE SHAM MICE. THESE DIFFERENCES WERE ASSOCIATED WITH HIGH DATA VARIABILITY IN THE SHAM GROUP, WHILE IN THE RUPP GROUP, PERFUSION LOOKS ?STIFFER.? DATA ALSO SUGGEST SEXDIMORPHISM IN THE VASCULAR RESPONSE SINCE FEMALE PUPS IN THE SHAM GROUP BUT NOT MALE PUPS SHOWED STATISTICALLY SIGNIFICANT DIFFERENCES IN RESPONSE TO THE WARM STIMULUS. AGAIN, THIS SEX-RELATED DIFFERENCE WAS ABSENT IN PUPS OF RUPP MICE. IN CONCLUSION, WE PRESENT A ROBUST QUANTITATIVE APPROACH FOR LSCI MEASUREMENTS THAT REVEALED ANOMALIES IN THE BRAIN BLOOD FLOW IN OFFSPRING OF THE RUPP MODEL OF PE.

OBJECTIVE: EITHER OBESITY OR VOCAL LOADING TASK LEADS TO ELEVATION OF PROINFLAMMATORY CYTOKINES SUCH AS INTERLEUKIN 6 (IL-6). HOWEVER, IT IS UNKNOWN WHETHER VOCAL PARAMETERS AFTER VOCAL LOADING ARE CORRELATED WITH BODY MASS INDEX (BMI) OR IL-6. WE HYPOTHESIZE THAT VOCAL LOADING INDUCES AN ELEVATION OF ACOUSTIC PARAMETERS OF VOICE AND SALIVARY IL-6 IN OVERWEIGHT AND OBESE PEOPLE. METHODS: A TOTAL OF 33 SCHOOLTEACHERS WITHOUT ANY SELF-REPORTED VOICE ALTERATIONS WERE INVITED TO PARTICIPATE IN THIS STUDY. PARTICIPANTS WERE CLASSIFIED ACCORDING TO BMI INTO NORMAL, OVERWEIGHT, AND OBESE GROUPS. THE VOCAL LOADING TASK CONSISTED OF LOUD SPEECH (60-90 MINUTES) IN THEIR CLASSROOM. SALIVARY AND VOICE SAMPLES WERE TAKEN BEFORE AND AFTER VOCAL LOADING. PERCEPTUAL AND SELF-REPORTED VOICE ALTERATIONS AND OBJECTIVE VOICE ANALYSES WERE INVESTIGATED. THE RELATIVE CONCENTRATION OF SALIVARY IL-6 WAS ESTIMATED BY A COLORIMETRIC ASSAY. RESULTS: OBESE TEACHERS SHOWED A SIGNIFICANT ELEVATION IN FUNDAMENTAL FREQUENCY VALUE AFTER VOCAL LOADING. IN ADDITION, REDUCTION IN HARMONICS-TO-NOISE RATIO (HNR) WAS OBSERVED IN TEACHERS WITH NORMAL WEIGHT AFTER VOCAL LOADING BUT NOT IN OVERWEIGHT OR OBESE GROUPS. NO SIGNIFICANT CORRELATION WAS OBSERVED BETWEEN BMI AND ANY OF THE ACOUSTIC PARAMETERS ANALYZED OR SALIVARY IL-6 LEVELS. FURTHERMORE, TEACHERS WHO WERE OVERWEIGHT SHOWED A SIGNIFICANT INCREASE IN THE SALIVARY IL-6 LEVELS AFTER VOCAL LOADING. INTERESTINGLY, SALIVARY IL-6 LEVELS WERE POSITIVELY CORRELATED WITH HNR VALUE IN THE OVERWEIGHT GROUP AFTER VOCAL LOADING. CONCLUSION: EXCESSIVE BODY WEIGHT IS RELATED TO ALTERATIONS IN FUNDAMENTAL FREQUENCY AND HNR. IN ADDITION, HNR, BUT NOT BMI, IS ASSOCIATED WITH SALIVARY IL-6 LEVELS IN OVERWEIGHT TEACHERS.

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.

MUCH EPIDEMIOLOGICAL EVIDENCE HAS SHOWN THAT THE OFFSPRING BORN TO PREECLAMPTIC WOMEN (HYPERTENSION IN PREGNANCY) HAVE A HIGH RISK FOR DEVELOPING CARDIOVASCULAR DISEASES AND NEUROCOGNITIVE DISORDERS, INCLUDING LANGUAGE DISORDERS LATER IN LIFE. NEVERTHELESS, THE ANALYSIS OF LANGUAGE DISORDERS IN CHILDREN BORN TO PREECLAMPTIC WOMEN IS LIMITED. THEREFORE, THE PRESENT STUDY AIMS TO INVESTIGATE ORAL LANGUAGE IN CHILDREN OF MOTHERS WHO HAD PREECLAMPSIA. METHOD: THIS IS A CROSS-SECTIONAL STUDY, PREVIOUSLY APPROVED BY THE SCIENTIFIC ETHICS COMMITTEE OF THE HOSPITAL CLÍNICO HERMINDA MARTIN OF CHILLÁN, CHILE. THE ANALYSIS WAS PERFORMED ON A THIRD (N = 17) OF A TARGET UNIVERSE OF 56 CHILDREN. THESE WERE SUB-DIVIDED INTO GROUPS OF PRE-SCHOOL CHILDREN BORN TO NORMAL PREGNANCIES (N = 4) AND PREECLAMPTIC WOMEN (N = 3); AND CHILDREN OF SCHOOL AGE BORN TO NORMAL (N = 5) AND PREECLAMPTIC WOMEN (N = 5). WE EVALUATED IN GENERAL (SCREENING) THE DIFFERENT LEVELS OF LANGUAGE AND NARRATIVE DISCOURSE USING AN ADAPTATION OF THE TESTS AVAILABLE IN THE LITERATURE. RESULTS: NO STATISTICALLY SIGNIFICANT DIFFERENCES BETWEEN CHILDREN OF PRE-SCHOOL OR SCHOOL AGE FROM BOTH GROUPS OF PREGNANCIES WERE FOUND IN THE TOTAL SCORE OF THE LANGUAGE EVALUATION TEST. HOWEVER, IN THE MORE DETAILED ANALYSIS OF EITHER THE EXPRESSIVE OR THE COMPREHENSIVE ASPECT, SPECIFIC DIFFERENCES WERE DETECTED THAT RESULTED IN A LOW PERCENTAGE OF CHILDREN BORN TO MOTHERS WITH PRE-ECLAMPSIA IN THE PRE-SCHOOL STAGE WHO FAILED TO UNDERSTAND OR EXPRESS PART OF THE DELIVERED MESSAGE (END OF A TALE) COMPARED TO THE RESPECTIVE CONTROLS. CONCLUSIONS: THE RESULTS INDICATE THAT CHILDREN BORN TO MOTHERS WITH PREECLAMPSIA MAY HAVE DECREASED UNDERSTANDING AND EXPRESSION OF NARRATIVE DISCOURSE AT PRESCHOOL AGE. WE SUGGEST THAT PREECLAMPSIA MIGHT CAUSE DIMINISHED ORAL VERBAL ABILITY IN OFFSPRING.

EVIDENCE FROM CLINICAL STUDIES HAS PROPOSED THAT CHILDREN BORN FROM PREECLAMPTIC WOMEN HAVE A HIGHER RISK OF SUFFERING NEUROLOGICAL, PSYCHOLOGICAL, OR BEHAVIORAL ALTERATIONS. HOWEVER, TO DATE, THE MECHANISMS BEHIND THESE OUTCOMES ARE POORLY UNDERSTOOD. HERE, WE SPECULATE THAT THE NEURODEVELOPMENTAL ALTERATIONS IN THE CHILDREN OF PREECLAMPTIC PREGNANCIES RESULT FROM IMPAIRED ANGIOGENESIS. THE PRO-ANGIOGENIC FACTORS VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND PLACENTAL GROWTH FACTOR (PLGF) ARE KEY REGULATORS OF BOTH VASCULAR AND NEUROLOGICAL DEVELOPMENT, AND IT HAS BEEN WIDELY DEMONSTRATED THAT UMBILICAL BLOOD OF PREECLAMPTIC PREGNANCIES CONTAINS HIGH LEVELS OF SOLUBLE VEGF RECEPTOR TYPE 1 (SFLT-1), A DECOY RECEPTOR OF VEGF. AS A CONSEQUENCE, THIS ANTI-ANGIOGENIC STATE COULD LEAD TO LONG-LASTING NEUROLOGICAL OUTCOMES. IN THIS NON-SYSTEMATIC REVIEW, WE PROPOSE THAT ALTERATIONS IN THE CIRCULATING CONCENTRATIONS OF VEGF, PLGF, AND SFLT-1 IN PREECLAMPTIC PREGNANCIES WILL AFFECT BOTH FETAL CEREBROVASCULAR FUNCTION AND NEURODEVELOPMENT, WHICH IN TURN MAY CAUSE COGNITIVE ALTERATIONS IN POST-NATAL LIFE.

CEREBRAL COMPLICATIONS IN PREECLAMPSIA CONTRIBUTE SUBSTANTIALLY TO MATERNAL MORTALITY AND MORBIDITY. THERE IS A LACK OF RELIABLE AND ACCESSIBLE PREDICTORS FOR PREECLAMPSIA-RELATED CEREBRAL COMPLICATIONS. IN THIS STUDY, PLASMA FROM WOMEN WITH PREECLAMPSIA (N = 28), WOMEN WITH NORMAL PREGNANCIES (N = 28) AND NON-PREGNANT WOMEN (N = 16) WAS ANALYZED FOR CONCENTRATIONS OF THE CEREBRAL BIOMARKERS NEUROFILAMENT LIGHT (NFL), TAU, NEURON-SPECIFIC ENOLASE (NSE) AND S100B. THEN, AN IN VITRO BLOOD-BRAIN BARRIER (BBB) MODEL, BASED ON THE HUMAN CEREBRAL MICROVASCULAR ENDOTHELIAL CELL LINE (HCMEC/D3), WAS EMPLOYED TO ASSESS THE EFFECT OF PLASMA FROM THE THREE STUDY GROUPS. TRANSENDOTHELIAL ELECTRICAL RESISTANCE (TEER) WAS USED AS AN ESTIMATION OF BBB INTEGRITY. NFL AND TAU ARE PROTEINS EXPRESSED IN AXONS, NSE IN NEURONS AND S100B IN GLIAL CELLS AND ARE USED AS BIOMARKERS FOR NEUROLOGICAL INJURY IN OTHER DISEASES SUCH AS DEMENTIA, TRAUMATIC BRAIN INJURY AND HYPOXIC BRAIN INJURY. PLASMA CONCENTRATIONS OF NFL, TAU, NSE AND S100B WERE ALL HIGHER IN WOMEN WITH PREECLAMPSIA COMPARED WITH WOMEN WITH NORMAL PREGNANCIES (8.85 VS. 5.25 NG/L, P < 0.001; 2.90 VS. 2.40 NG/L, P < 0.05; 3.50 VS. 2.37 ?G/L, P < 0.001 AND 0.08 VS. 0.05 ?G/L, P < 0.01, RESPECTIVELY). PLASMA CONCENTRATIONS OF NFL WERE ALSO HIGHER IN WOMEN WITH PREECLAMPSIA COMPARED WITH NON-PREGNANT WOMEN (P < 0.001). HIGHER PLASMA CONCENTRATIONS OF THE CEREBRAL BIOMARKER NFL WERE ASSOCIATED WITH DECREASED TEER (P = 0.002) IN AN IN VITRO MODEL OF THE BBB, A FINDING WHICH INDICATES THAT NFL COULD BE A PROMISING BIOMARKER FOR BBB ALTERATIONS IN PREECLAMPSIA.

PREECLAMPSIA, A PREGNANCY-RELATED ENDOTHELIAL DISORDER, IS ASSOCIATED WITH BOTH CARDIOVASCULAR AND CEREBROVASCULAR COMPLICATIONS. PREECLAMPSIA REQUIRES THE PRESENCE OF A PLACENTA AS PART OF ITS PATHOPHYSIOLOGY, YET THE ROLE OF THIS ORGAN IN THE CEREBROVASCULAR COMPLICATIONS REMAINS UNCLEAR. RESEARCH HAS SHOWN THAT CIRCULATING SMALL EXTRACELLULAR VESICLES (ALSO KNOWN AS EXOSOMES) PRESENT IN PREECLAMPSIA PLASMA CAN GENERATE ENDOTHELIAL DYSFUNCTION, BUT IT IS UNCLEAR WHETHER THE IMPAIRMENT OF FUNCTION OF BRAIN ENDOTHELIAL CELLS AT THE BLOOD-BRAIN BARRIER IS SECONDARY TO PLASMA-DERIVED OR PLACENTAL-DERIVED EXOSOMES. IN THIS STUDY, WE EVALUATED THE EFFECT OF SMALL EXTRACELLULAR VESICLES ISOLATED FROM PLASMA SAMPLES OF WOMEN WITH PREECLAMPSIA (N=12) AND WOMEN WITH NORMAL PREGNANCY (N=11) AS WELL AS FROM HUMAN PLACENTAL EXPLANTS FROM NORMOTENSIVE PREGNANCIES (N=6) SUBJECTED TO HYPOXIA (1% OXYGEN) ON THE INTEGRITY OF THE BLOOD-BRAIN BARRIER, USING BOTH IN VITRO AND ANIMAL MODELS. EXPOSURE OF HUMAN-DERIVED BRAIN ENDOTHELIAL CELL MONOLAYERS TO PLASMA AND PLASMA-DERIVED SMALL EXTRACELLULAR VESICLES FROM PREECLAMPTIC PREGNANCIES INCREASED THE PERMEABILITY AND REDUCED THE TRANSENDOTHELIAL ELECTRICAL RESISTANCE. A SIMILAR OUTCOME WAS OBSERVED WITH HYPOXIC PLACENTAL-DERIVED SMALL EXTRACELLULAR VESICLES, WHICH ALSO INCREASED THE PERMEABILITY TO EVAN?S BLUE IN THE BRAIN OF C57BL6 NONPREGNANT MICE. COTREATMENT WITH MAGNESIUM SULFATE REVERSED THE EFFECTS ELICITED BY PLASMA, PLASMA-DERIVED, AND HYPOXIC PLACENTAL-DERIVED SMALL EXTRACELLULAR VESICLES IN THE EMPLOYED MODELS. THUS, CIRCULATING SMALL EXTRACELLULAR VESICLES IN PLASMA FROM WOMEN WITH PREECLAMPSIA OR FROM HYPOXIC PLACENTAE DISRUPT THE BLOOD-BRAIN BARRIER, WHICH CAN BE PREVENTED USING MAGNESIUM SULFATE. THESE FINDINGS PROVIDE NEW INSIGHTS INTO THE PATHOPHYSIOLOGY OF CEREBRAL COMPLICATIONS ASSOCIATED WITH PREECLAMPSIA.

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.

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

INTRODUCTION PLACENTAS FROM BOTH EARLY-ONSET (EOPE) AND LATE-ONSET PRE-ECLAMPSIA (LOPE) EXHIBIT SIGNS OF UNDERPERFUSION, WHICH IN TURN, MAY BE ASSOCIATED WITH ALTERED ANGIOGENESIS. TYROSINE 951 (Y951) AND Y1175 PHOSPHORYLATION OF THE VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 (VEGFR2) INDUCED BY VEGF TRIGGERS THE ANGIOGENESIS PROCESS. ENDOTHELIAL MARKERS SUCH AS CD31 AND CD34 HAVE BEEN USED FOR ESTIMATING ANGIOGENIC PROCESSES IN SEVERAL TISSUES, INCLUDING PLACENTA. WE ASKED WHETHER VASCULAR DENSITY IN PLACENTAL VILLI WAS RELATED TO Y951/Y1175 PHOSPHORYLATION OF VEGFR2 IN LOPE OR EOPE. METHODS WE OBTAINED PLACENTAL SAMPLES FROM WOMEN WITH NORMAL PREGNANCIES (N = 22), LOPE (N = 13), EOPE (N = 15) AND PRETERM DELIVERIES (N = 10). SLICES FROM PLACENTAL TISSUE WERE USED FOR CD31 IMMUNOSTAINING. WE ESTIMATED THE EXPRESSION OF CD31, CD34, VEGF, AND VEGFR2 BY WESTERN BLOT AND QUANTITATIVE PCR. Y951 PHOSPHORYLATION OF VEGFR2 WAS ESTIMATED BY WESTERN BLOT, WHEREAS Y1175 PHOSPHORYLATION WAS ANALYZED BY ELISA. RESULTS VESSEL DENSITY IN TERMINAL VILLI AND CD31 AND CD34 PROTEIN ABUNDANCE WERE INCREASED IN LOPE AND EOPE COMPARED TO NORMAL PREGNANCY. HOWEVER, MRNA LEVELS FOR CD31 AND CD34 WERE LOWER IN LOPE THAN IN NORMAL PREGNANCY AND VEGF MRNA WAS HIGHER IN EOPE. VEGFR2 PROTEIN CONCENTRATION WAS NOT DIFFERENT AMONG THE STUDIED GROUPS. Y951 AND Y1175 PHOSPHORYLATION OF VEGFR2 WAS HIGHER IN LOPE THAN IN THE NORMOTENSIVE GROUP, BUT ONLY Y951 EXHIBITED GREATER PHOSPHORYLATION IN EOPE COMPARED TO NORMAL PREGNANCY.

FUNCTIONAL FOODS ARE ALIMENTS THAT ARE THOUGHT TO HAVE PHYSIOLOGICAL BENEFITS AND/OR REDUCE THE RISK OF CHRONIC DISEASE, BEYOND THEIR BASIC NUTRITIONAL FUNCTIONS, WHICH COULD BE PRODUCED BY ADDING NEW INGREDIENTS OR MORE OF EXISTING INGREDIENTS USING FOOD ENGINEERING TECHNIQUES. IN THIS REGARD, INCREASING HEALTH CONSCIOUSNESS WORLDWIDE HAS BEEN ONE OF THE MOST STIMULATING FACTORS FOR RAPID GLOBAL GROWTH OF THE FUNCTIONAL FOOD INDUSTRY (HASLER, J AM COLL NUTR 19:499S?506S, 2000). THEN, DEVELOPMENT OF FUNCTIONAL FOOD PRODUCTS IS A GROWING RESEARCH NICHE WORLDWIDE.

OBJECTIVES: TO COMPARE THE NEUROVASCULAR STRUCTURE AND PROTEIN EXPRESSION OF CEREBRAL ANGIOGENESIS MARKERS AS WELL AS MORPHOMETRIC AND HISTOLOGICAL VARIATIONS IN THE BRAIN OF CHILDREN OF MOTHERS WITH AND WITHOUT HYPERTENSIVE DISORDER OF PREGNANCY. METHODS: AN ANALYTICAL OBSERVATIONAL STUDY OF CASES AND CONTROLS, RETROSPECTIVE, WITH DOUBLE, RANDOM, AND BLINDED READING OF HISTOLOGICAL SAMPLES, OF BRAINS FROM THE PRODUCTS OF PREGNANCIES WITH AND WITHOUT HYPERTENSIVE DISEASES OF PREGNANCY THAT REST IN THE ARCHIVES OF THE HOSPITAL UNIVERSITARIO SAN IGNACIO. RESULTS: THE PROJECT IS ONGOING. TO DATE, THE HISTOLOGICAL STRUCTURE OF 17 CASES HAS BEEN REVIEWED IN HEMATOXYLIN-EOSIN. THE PRESENCE OF RED NEURONS, AREAS OF HEMORRHAGE, AREAS OF RECENT AND OLD INFARCTION AS WELL AS THE PRESENCE OF CALCIFICATIONS IN THE CORTEX, HIPPOCAMPUS, AND CEREBELLUM HAVE BEEN LOOKED FOR AND DOCUMENTED. CONCLUSION: OUR KNOWLEDGE OF THE PREECLAMPSIA IMPACT ON THE DEVELOPMENT OF THE FETUS IS STILL LIMITED. STUDIES LIKE OURS WANT TO HELP TO UNDERSTAND THE INTRAUTERINE ENVIRONMENT AND ITS CONSEQUENCES.

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.

BACKGROUND: CEREBRAL COMPLICATIONS IN PREECLAMPSIA ARE LEADING CAUSES OF MATERNAL MORTALITY WORLDWIDE BUT PATHOPHYSIOLOGY IS LARGELY UNKNOWN AND A CHALLENGE TO STUDY. USING AN IN VITRO MODEL OF THE HUMAN BLOOD-BRAIN BARRIER (BBB), WE EXPLORED THE ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 (VEGFR2) IN PREECLAMPSIA. METHODS: THE HUMAN BRAIN ENDOTHELIAL CELL LINE (HCMEC/D3) CULTURED ON TRANWELLS INSERT WAS EXPOSED (12 HOURS) TO PLASMA FROM WOMEN WITH PREECLAMPSIA (N = 28), NORMAL PREGNANCY (N = 28), AND NONPREGNANT (N = 16) CONTROLS. TRANSENDOTHELIAL ELECTRICAL RESISTANCE (TEER) AND PERMEABILITY TO 70 KDA FLUORESCEIN ISOTHIOCYANATE (FITC)-DEXTRAN WERE MEASURED FOR THE ASSESSMENT OF BBB INTEGRITY. WE EXPLORED POSSIBLE UNDERLYING MECHANISMS, WITH A FOCUS ON THE EXPRESSION OF TIGHT JUNCTION PROTEINS AND PHOSPHORYLATION OF 2 TYROSINE RESIDUES OF VEGFR2, ASSOCIATED WITH VASCULAR PERMEABILITY AND MIGRATION (PY951) AND CELL PROLIFERATION (PY1175). PLASMA CONCENTRATIONS OF SOLUBLE FMS-LIKE TYROSINE KINASE-1 (SFLT-1) AND PLACENTAL GROWTH FACTOR (PLGF) WERE ALSO MEASURED. RESULTS: HCMEC/D3 EXPOSED TO PLASMA FROM WOMEN WITH PREECLAMPSIA EXHIBITED REDUCED TEER AND INCREASED PERMEABILITY TO 70 KDA FITC-DEXTRAN. THESE CELLS UPREGULATED THE MESSENGER RIBONUCLEIC ACID (MRNA) LEVELS OF VEGFR2, AND PY951-VEGFR2, BUT REDUCED PY1175-VEGFR2 (P < 0.05 IN ALL CASES). NO DIFFERENCE IN MRNA EXPRESSION OF TIGHT JUNCTION PROTEIN WAS OBSERVED BETWEEN GROUPS. THERE WAS NO CORRELATION BETWEEN ANGIOGENIC BIOMARKERS AND BBB PERMEABILITY. CONCLUSIONS: WE PRESENT A PROMISING IN VITRO MODEL OF THE BBB IN PREECLAMPSIA. SELECTIVE TYROSINE PHOSPHORYLATION OF VEGFR2 MAY PARTICIPATE IN THE INCREASED BBB PERMEABILITY IN PREECLAMPSIA IRRESPECTIVE OF PLASMA CONCENTRATIONS OF ANGIOGENIC BIOMARKERS.

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.