Compromised barrier integrity of human feto-placental vessels from gestational diabetic pregnancies is related to downregulation of occludin expression

© 2020, The Author(s). Aims/hypothesis: Reduced occupancy of junctional occludin is a feature of human placental vessels in the diabetic milieu. However, the functional consequence of this and whether this loss is due to differential expression of occludin splice variants is not known. Our study aimed to investigate the effects of gestational diabetes mellitus (GDM), and its treatment, on endothelial junctional integrity, gene and protein expression of occludin splice variants, and potential regulation of expression by microRNAs (miRNAs). Methods: Term placentas were obtained from normal pregnancies (n = 21), and pregnancies complicated by GDM where glucose levels were controlled by diet (n = 11) or metformin (n = 6). Gene and microRNA (miRNA) expression were determined by quantitative real-time PCR; protein expression by immunoblotting; endothelial junctional occupancy by fluorescence microscopy and systematic sampling; and paracellular leakage by perfusion of placental microvascular beds with 76 Mr dextran. Transfection studies of miRNAs that target OCLN were performed in HUVECs, and the trans-endothelial electrical resistance and tracer permeability of the HUVECs were measured. Results: All three predicted OCLN gene splice variants and two occludin protein isoforms were found in human placental samples. In placental samples from diet-controlled GDM (d-GDM) pregnancies we found a lower percentage of conduit vessels showing occludin immunoreactivity (12%, p < 0.01), decreased levels of the fully functional occludin isoform-A protein (29%), and differential gene expression of OCLN variant 2 (33% decrease), variant 3 (3.3-fold increase). These changes were not seen in samples from the group with metformin-controlled GDM. In d-GDM placentas, increased numbers of conduit microvessels demonstrated extravasation of 76 Mr dextran (2.0-fold). In d-GDM expression of one of the five potential miRNAs targeting OCLN, miR-181a-5p, expression was 2.1-fold that in normal pregnancies. Experimental overexpression of miR-181a-5p in HUVECs from normal pregnancies resulted in a highly significant downregulation of OCLN variant 1 (69%) and variant 2 (46%) gene expression, with decreased trans-endothelial resistance (78%) and increase in tracer permeability (1.3-fold). Conclusions/interpretation: Downregulation of expression of OCLN variant 2 and the fully functional occludin isoform-A protein are a feature of placentas in d-GDM pregnancies. These may be behind the loss of junctional occludin and the increased extravasation of exogenous dextran observed. miR-181a-5p was in part responsible for the downregulation of occludin in placentas from d-GDM pregnancies. Induced overexpression of miR-181a-5p compromised the integrity of the endothelial barrier. Our data suggest that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy may not be enough to prevent an alteration in the expression of occludin and the subsequent functional consequences in placentas and impaired vascular barrier function in offspring. [Figure not available: see fulltext.

The effect of gestational diabetes, and its treatment, on the expression of Occludin, VE-cadherin & Plakoglobin splice variants in the human placental endothelial barrier

The placenta is essential for the development, survival and growth of the embryo, as it forms the critical interface between the fetus and the mother. Gestational diabetes mellitus (GDM) is a type of diabetes typically detected during the second or third trimester of pregnancy. Reduced occupancy of tight and adherens junctional molecules (Occludin and VE-cadherin) and disrupted junctions are a feature of human placental vessels under a diabetic milieu. Differential expression of gene splice variants and resultant protein isoforms may be behind the changes in junctional molecules and was the central hypothesis of this PhD study. The role of angiogenic growth factors and epigenetic control, if any, were also investigated in order to elucidate mechanisms behind gene expression changes. Human placental tissue from normal pregnancies were compared with those complicated by GDM where glucose levels were treated by a change of diet or by medication (metformin). Placental and umbilical cord tissue was collected from 40 term singleton pregnancies delivered by caesarean section. Gene expression was determined by qPCR; protein expression by immunoblotting, and junctional occupancy by the systematic sampling of immuno-positive vessels. Diet-controlled GDM placental samples showed differential expression of Occludin gene and protein: Occludin variant 2 was reduced (1.5-fold), while Occludin variant 3 was increased (3.3-fold) compared to normal. Occludin isoform-A levels (fully functional isoform) were decreased (1.4-fold), and lower percentage of vessels showed junctional Occludin (12%) compared to normal. Additionally, VE-cadherin gene and protein expression remained unchanged between normal and GDM placental samples. Finally, Plakoglobin gene expression was higher in diet-controlled GDM samples (1.6-fold) compared to normal. In metformin-controlled GDM placental samples, Occludin, VE-cadherin and Plakoglobin gene and protein expression were similar to normal placental samples. We explored if differential expression of angiogenic factors was behind the downregulation of Occludin found in GDM placentae. Assessment of the gene expression of angiogenic/permeability factors (VEGF-A, Ang-1, Ang-2 and MMP-9) with their receptors (VEGFR-2 and Tie-2) revealed similar expression of these factors between normal and GDM placental samples (p > 0.05). Total VEGF-A protein expression was downregulated (1.3-fold) in diet-controlled GDM placental samples compared to normal. An ex vivo model (placental chorionic explants cultured under hyperglycaemic conditions (15 mM D-glucose) for 3-hours (n= 4), or 24-hours (n= 4)) showed that hyperglycaemia did not cause downregulation of Occludin gene expression or junctional localisation in the placental vasculature compared to control (p > 0.05). To determine the mechanism behind the changes in Occludin expression, our study examined whether GDM affected the expression of epigenetic markers in placenta. Quantification of the DNA methylation levels of Occludin promoter showed no significant change between normal and GDM placental pregnancies. Additionally, miR181a-5p expression was higher (2.1-fold) in diet-controlled GDM placental samples compared to normal. To confirm the effects of miR-181a-5p in Occludin gene expression, miR-181a-5p was overexpressed in HUVEC. Results showed downregulation of Occludin variant 1 (3.2-fold) and variant 2 (1.8-fold) gene expression in the presence of miR181a-5p. In conclusion, our study showed that the junctional molecules Occludin, VE-cadherin and Plakoglobin presented several gene splice variants and protein isoforms in human placental samples. Altered gene expression of Occludin and Plakoglobin were found in diet-controlled GDM placentae. Downregulation of Occludin was in part the result of overexpression of miR-181a-5p. The stable expression of the target genes in the metformin-controlled GDM group may not be a result of the corrective effects of metformin upon glucose. Our data suggests that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy (diet-controlled) may not be enough to prevent an alteration in the expression of Occludin

The effect of gestational diabetes, and its treatment, on the expression of Occludin, VE-cadherin & Plakoglobin splice variants in the human placental endothelial barrier

The placenta is essential for the development, survival and growth of the embryo, as it forms the critical interface between the fetus and the mother. Gestational diabetes mellitus (GDM) is a type of diabetes typically detected during the second or third trimester of pregnancy. Reduced occupancy of tight and adherens junctional molecules (Occludin and VE-cadherin) and disrupted junctions are a feature of human placental vessels under a diabetic milieu. Differential expression of gene splice variants and resultant protein isoforms may be behind the changes in junctional molecules and was the central hypothesis of this PhD study. The role of angiogenic growth factors and epigenetic control, if any, were also investigated in order to elucidate mechanisms behind gene expression changes. Human placental tissue from normal pregnancies were compared with those complicated by GDM where glucose levels were treated by a change of diet or by medication (metformin). Placental and umbilical cord tissue was collected from 40 term singleton pregnancies delivered by caesarean section. Gene expression was determined by qPCR; protein expression by immunoblotting, and junctional occupancy by the systematic sampling of immuno-positive vessels. Diet-controlled GDM placental samples showed differential expression of Occludin gene and protein: Occludin variant 2 was reduced (1.5-fold), while Occludin variant 3 was increased (3.3-fold) compared to normal. Occludin isoform-A levels (fully functional isoform) were decreased (1.4-fold), and lower percentage of vessels showed junctional Occludin (12%) compared to normal. Additionally, VE-cadherin gene and protein expression remained unchanged between normal and GDM placental samples. Finally, Plakoglobin gene expression was higher in diet-controlled GDM samples (1.6-fold) compared to normal. In metformin-controlled GDM placental samples, Occludin, VE-cadherin and Plakoglobin gene and protein expression were similar to normal placental samples. \ud We explored if differential expression of angiogenic factors was behind the downregulation of Occludin found in GDM placentae. Assessment of the gene expression of angiogenic/permeability factors (VEGF-A, Ang-1, Ang-2 and MMP-9) with their receptors (VEGFR-2 and Tie-2) revealed similar expression of these factors between normal and GDM placental samples (p > 0.05). Total VEGF-A protein expression was downregulated (1.3-fold) in diet-controlled GDM placental samples compared to normal. An ex vivo model (placental chorionic explants cultured under hyperglycaemic conditions (15 mM D-glucose) for 3-hours (n= 4), or 24-hours (n= 4)) showed that hyperglycaemia did not cause downregulation of Occludin gene expression or junctional localisation in the placental vasculature compared to control (p > 0.05). To determine the mechanism behind the changes in Occludin expression, our study examined whether GDM affected the expression of epigenetic markers in placenta. Quantification of the DNA methylation levels of Occludin promoter showed no significant change between normal and GDM placental pregnancies. Additionally, miR181a-5p expression was higher (2.1-fold) in diet-controlled GDM placental samples compared to normal. To confirm the effects of miR-181a-5p in Occludin gene expression, miR-181a-5p was overexpressed in HUVEC. Results showed downregulation of Occludin variant 1 (3.2-fold) and variant 2 (1.8-fold) gene expression in the presence of miR181a-5p. In conclusion, our study showed that the junctional molecules Occludin, VE-cadherin and Plakoglobin presented several gene splice variants and protein isoforms in human placental samples. Altered gene expression of Occludin and Plakoglobin were found in diet-controlled GDM placentae. Downregulation of Occludin was in part the result of overexpression of miR-181a-5p. The stable expression of the target genes in the metformin-controlled GDM group may not be a result of the corrective effects of metformin upon glucose. Our data suggests that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy (diet-controlled) may not be enough to prevent an alteration in the expression of Occludin

Establishment, Genetic Diversity, and Habitat Suitability of <i>Aedes albopictus</i> Populations from Ecuador

Aedes albopictus, also known as the tiger mosquito, is widespread worldwide across tropical, subtropical, and temperate regions. This insect is associated with the transmission of several vector-borne diseases, and, as such, monitoring its distribution is highly important for public health. In Ecuador, Ae. albopictus was first reported in 2017 in Guayaquil. Since then, the vector has been identified in the Northeastern lowlands and the Amazon basin. This study aims to determine the genetic diversity of Ecuadorian populations of Ae. albopictus through the analysis of the mitochondrial gene COI and to describe the potential distribution areas of this species within the country. The genetic diversity was determined by combining phylogenetic and population genetics analyses of five localities in Ecuador. Results showed two haplotypes in the Ecuadorian populations of Ae. albopictus. Haplotype 1 (H1) was found in the coastal and Amazon individuals, while haplotype 2 (H2) was only found in the three northeastern lowlands sites. In a worldwide context, H1 is the most widespread in 21 countries with temperate and tropical habitats. In contrast, H2 distribution is limited to five countries in tropical regions, suggesting fewer adaptation traits. Our prediction model showed a suitable habitat for Ae. albopictus in all regions (coastal, Amazon basin, and Andean lowland regions and the Galápagos Islands) of Ecuador. Hence, understanding different aspects of the vector can help us implement better control strategies for surveillance and vectorial control in Ecuador

Two Haplotypes of Aedes aegypti Detected by ND4 Mitochondrial Marker in Three Regions of Ecuador

Aedes aegypti, also known as the yellow fever mosquito, is the main vector of several arboviruses. In Ecuador, dengue and chikungunya are the most prevalent mosquito-borne diseases. Hence, there is a need to understand the population dynamics and genetic structure of the vector in tropical areas for a better approach towards effective vector control programs. This study aimed to assess the genetic diversity of Ae. aegypti, through the analyses of the mitochondrial gene ND4, using a combination of phylogenetic and population genetic structure from 17 sites in Ecuador. Results showed two haplotypes in the Ecuadorian populations of Ae. aegypti. Haplotype 1 was closely related to Ae. aegypti reported from America, Asia, and West Africa. Haplotype 2 was only related to samples from America. The sampled vectors from the diverse localities showed low nucleotide diversity (π = 0–0.01685) and genetic differentiation (FST = 0.152). AMOVA analyses indicated that most of the variation (85–91%) occurred within populations, suggesting that geographical barriers have little effect on the genetic structure of Ecuadorian populations of Ae. aegypti. These results agree with the one main population (K = 1) detected by Structure. Vector genetic identity may be a key factor in the planning of vector control strategies

Two Haplotypes of <i>Aedes aegypti</i> Detected by ND4 Mitochondrial Marker in Three Regions of Ecuador

Aedes aegypti, also known as the yellow fever mosquito, is the main vector of several arboviruses. In Ecuador, dengue and chikungunya are the most prevalent mosquito-borne diseases. Hence, there is a need to understand the population dynamics and genetic structure of the vector in tropical areas for a better approach towards effective vector control programs. This study aimed to assess the genetic diversity of Ae. aegypti, through the analyses of the mitochondrial gene ND4, using a combination of phylogenetic and population genetic structure from 17 sites in Ecuador. Results showed two haplotypes in the Ecuadorian populations of Ae. aegypti. Haplotype 1 was closely related to Ae. aegypti reported from America, Asia, and West Africa. Haplotype 2 was only related to samples from America. The sampled vectors from the diverse localities showed low nucleotide diversity (π = 0–0.01685) and genetic differentiation (FST = 0.152). AMOVA analyses indicated that most of the variation (85–91%) occurred within populations, suggesting that geographical barriers have little effect on the genetic structure of Ecuadorian populations of Ae. aegypti. These results agree with the one main population (K = 1) detected by Structure. Vector genetic identity may be a key factor in the planning of vector control strategies

Determinants of Aedes mosquito density as an indicator of arbovirus transmission risk in three sites affected by co-circulation of globally spreading arboviruses in Colombia, Ecuador and Argentina

Background: The global impact of Zika virus in Latin America has drawn renewed attention to circulating mosquito-borne viruses in this region, such as dengue and chikungunya. Our objective was to assess socio-ecological factors associated with Aedes mosquito vector density as a measure of arbovirus transmission risk in three cities of potentially recent Zika virus introduction: Ibagué, Colombia; Manta, Ecuador; and Posadas, Argentina, in order to inform disease mitigation strategies. Methods: We sampled Aedes mosquito populations in a total of 1086 households, using indoor and peridomestic mosquito collection methods, including light traps, resting traps, traps equipped with chemical attractant and aspirators. For each sampled household, we collected socio-economic data using structured questionnaires and data on microenvironmental conditions using iButton data loggers. Results: A total of 3230 female Aedes mosquitoes were collected, of which 99.8% were Aedes aegypti and 0.2% were Aedes albopictus. Mean female Aedes mosquito density per household was 1.71 (standard deviation: 2.84). We used mixed-effects generalized linear Poisson regression analyses to identify predictors of Aedes density, using month, neighborhood and country as random-effects variables. Across study sites, the number of household occupants [incidence rate ratio (IRR): 1.08, 95% confidence interval (CI): 1.01–1.14], presence of entry points for mosquitoes into the household (IRR: 1.51, 95% CI: 1.30–1.76) and presence of decorative vegetation (IRR: 1.52, 95% CI: 1.22–1.88) were associated with higher Aedes density; while being in the highest wealth tertile of household wealth (IRR: 0.78, 95% CI: 0.66–0.92), knowledge of how arboviruses are transmitted (IRR: 0.94, 95% CI: 0.89–1.00) and regular emptying of water containers by occupants (IRR: 0.79, 95% CI: 0.67–0.92) were associated with lower Aedes density. Conclusions: Our study addresses the complexities of arbovirus vectors of global significance at the interface between human and mosquito populations. Our results point to several predictors of Aedes mosquito vector density in countries with co-circulation of multiple Aedes-borne viruses, and point to modifiable risk factors that may be useful for disease prevention and control.Fil: Talbot, Benoit. University of Ottawa; CanadáFil: Sander, Beate. University of Toronto; CanadáFil: Cevallos, Varsovia. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: González, Camila. Universidad de los Andes; ColombiaFil: Benítez, Denisse. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: Carissimo, Claudio. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Carrasquilla Ferro, María C.. Universidad de los Andes; ColombiaFil: Gauto, Neris. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Litwiñiuk, Sergio Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; ArgentinaFil: López, Karen. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Ortiz, Mario I.. Universidad de los Andes; ColombiaFil: Ponce, Patricio. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: Villota, Stephany D.. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: Zelaya, Fabian. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Espinel, Mauricio. Universidad Tecnica de Manabi; EcuadorFil: Wu, Jianhong. University of York; Reino UnidoFil: Miretti, Marcos Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; ArgentinaFil: Kulkarni, Manisha A.. University of Ottawa; Canad

Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application.

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human β-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2