Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

Experimental Investigation of Calcium-Doped Zinc Aluminate Nanoparticles as a Promising Material for Microwave Applications.

Miniaturization of microstrip patch antennas (MPAs) is vital in applications such as wireless networks, mobile devices, global positioning satellites, and upcoming wireless terminals. This miniaturization has led to a demand for new materials with higher permittivity compared to the existing ones. Zinc aluminate (ZnAl2O4) ceramic is an exceptional and versatile material in this context, thanks to its high dielectric permittivity and low tangent loss, making it suitable for microwave applications. This article explores the feasibility of sol-gel-prepared Ca-doped ZnAl2O4 ceramic nanoparticles to be useful in fabricating a MPA. These nanoparticles were examined using X-ray diffraction, which confirmed their polycrystalline structure, and the morphological investigation evidenced the spherical grains having a mean diameter of 16 nm. The dielectric permittivity of the ZnAl2O4Ca nanoparticles is 21.11, with a tangential loss of 0.0247. A prototype MPA made by using Ca-doped ZnAl2O4 nanoparticles showed a return loss of -20.92 dB at a resonance frequency of 6.8 GHz with a bandwidth of 600 MHz. These results indicate that Ca-doped ZnAl2O4 ceramic nanoparticles possess exceptional dielectric characteristics, which make them a promising candidate for MPA applications.

Transcriptomic and Functional Evidence That miRNA193a-3p Inhibits Lymphatic Endothelial Cell (LEC) and LEC + MCF-7 Spheroid Growth Directly and by Altering MCF-7 Secretome.

MicroRNA 193a-3p (miR193a-3p) is a short non-coding RNA with tumor suppressor properties. Breast cancer (BC) progression is governed by active interaction between breast cancer cells, vascular (V)/lymphatic (L) endothelial cells (ECs), and BC secretome. We have recently shown that miR193a-3p, a tumor suppressor miRNA, inhibits MCF-7 BC cell-driven growth of VECs via direct antimitogenic actions and alters MCF-7 secretome. Since LEC-BC cross-talk plays a key role in BC progression, we investigated the effects of miR193a-3p on MCF-7 secretome and estradiol-mediated growth effects in LECs and LEC + MCF-7 spheroids, and delineated the underlying mechanisms. Transfection of LECs with miR193a-3p, as well as secretome from MCF-7 transfected cells, inhibited LEC growth, and these effects were mimicked in LEC + MCF-7 spheroids. Moreover, miR193a-3p inhibited ERK1/2 and Akt phosphorylation in LECs and LEC + MCF-7 spheroids, which are importantly involved in promoting cancer development and metastasis. Treatment of LECs and LEC + MCF-7 spheroids with estradiol (E2)-induced growth, as well as ERK1/2 and Akt phosphorylation, and was abrogated by miR193a-3p and secretome from MCF-7 transfected cells. Gene expression analysis (GEA) in LEC + MCF-7 spheroids transfected with miR193a-3p showed significant upregulation of 54 genes and downregulation of 73 genes. Pathway enrichment analysis of regulated genes showed significant modulation of several pathways, including interferon, interleukin/cytokine-mediated signaling, innate immune system, ERK1/2 cascade, apoptosis, and estrogen receptor signaling. Transcriptomic analysis showed downregulation in interferon and anti-apoptotic and pro-growth molecules, such as IFI6, IFIT1, OSA1/2, IFITM1, HLA-A/B, PSMB8/9, and PARP9, which are known to regulate BC progression. The cytokine proteome array of miR193a-3p transfected MCF secretome and confirmed the upregulation of several growth inhibitory cytokines, including IFNγ, Il-1a, IL-1ra, IL-32, IL-33, IL-24, IL-27, cystatin, C-reactive protein, Fas ligand, MIG, and sTIM3. Moreover, miR193a-3p alters factors in MCF-7 secretome, which represses ERK1/2 and Akt phosphorylation, induces pro-apoptotic protein and apoptosis in LECs, and downregulates interferon-associated proteins known to promote cancer growth and metastasis. In conclusion, miR193a-3p can potentially modify the tumor microenvironment by altering pro-growth BC secretome and inhibiting LEC growth, and may represent a therapeutic molecule to target breast tumors/cancer.

Response of tropical trees to elevated Ozone: a Free Air Ozone Enrichment study.

Tropospheric ozone (O3) has become one of the main urban air pollutants. In the present study, we assessed impact of ambient and future ground-level O3 on nine commonly growing urban tree species under Free Air Ozone Enrichment (FAOE) condition. During the study period, mean ambient and elevated ozone (EO3) concentrations were 48.59 and 69.62 ppb, respectively. Under EO3 treatment, stomatal density (SD) significantly decreased and guard cell length (GCL) increased in Azadirachta indica, Bougainvillea spectabilis, Plumeria rubra, Saraca asoca and Tabernaemontana divaricata, while SD increased and GCL decreased in Ficus benghalensis and Terminalia arjuna. Proline levels increased in all the nine plant species under EO3 condition. EO3 significantly reduced photosynthetic rate, stomatal conductance (gs), and transpiration rates (E). Only A. indica and N. indicum showed higher gs and E under EO3 treatment. Water use efficiency (WUE) significantly increased in F. benghalensis and decreased in A. indica and T. divaricata. Air Pollution Tolerance Index (APTI) significantly increased in Ficus religiosa and S. asoca whereas it decreased in B. spectabilis and A. indica. Of all the plant species B. spectabilis and A. indica were the most sensitive to EO3 (high gs and less ascorbic acid content) while S. asoca and F. religiosa were the most tolerant (lowgs and more ascorbic acid content). The sensitivity of urban tree species to EO3 is a cause of concern and should be considered for future urban forestry programmes. Our study should guide more such studies to identify tolerant trees for urban air pollution abatement.

Micro-RNA193a-3p Inhibits Breast Cancer Cell Driven Growth of Vascular Endothelial Cells by Altering Secretome and Inhibiting Mitogenesis: Transcriptomic and Functional Evidence.

Breast cancer (BC) cell secretome in the tumor microenvironment (TME) facilitates neo-angiogenesis by promoting vascular endothelial cell (VEC) growth. Drugs that block BC cell growth or angiogenesis can restrict tumor growth and are of clinical relevance. Molecules that can target both BC cell and VEC growth as well as BC secretome may be more effective in treating BC. Since small non-coding microRNAs (miRs) regulate cell growth and miR193a-3p has onco-suppressor activity, we investigated whether miR193a-3p inhibits MCF-7-driven growth (proliferation, migration, capillary formation, signal transduction) of VECs. Using BC cells and VECs grown in monolayers or 3D spheroids and gene microarrays, we demonstrate that: pro-growth effects of MCF-7 and MDA-MB231 conditioned medium (CM) are lost in CM collected from MCF-7/MDA-MB231 cells pre-transfected with miR193a-3p (miR193a-CM). Moreover, miR193a-CM inhibited MAPK and Akt phosphorylation in VECs. In microarray gene expression studies, miR193a-CM upregulated 553 genes and downregulated 543 genes in VECs. Transcriptomic and pathway enrichment analysis of differentially regulated genes revealed downregulation of interferon-associated genes and pathways that induce angiogenesis and BC/tumor growth. An angiogenesis proteome array confirmed the downregulation of 20 pro-angiogenesis proteins by miR193a-CM in VECs. Additionally, in MCF-7 cells and VECs, estradiol (E2) downregulated miR193a-3p expression and induced growth. Ectopic expression of miR193a-3p abrogated the growth stimulatory effects of estradiol E2 and serum in MCF-7 cells and VECs, as well as in MCF-7 and MCF-7+VEC 3D spheroids. Immunostaining of MCF-7+VEC spheroid sections with ki67 showed miR193a-3p inhibits cell proliferation. Taken together, our findings provide first evidence that miR193a-3p abrogates MCF-7-driven growth of VECs by altering MCF-7 secretome and downregulating pro-growth interferon signals and proangiogenic proteins. Additionally, miR193a-3p inhibits serum and E2-induced growth of MCF-7, VECs, and MCF-7+VEC spheroids. In conclusion, miRNA193a-3p can potentially target/inhibit BC tumor angiogenesis via a dual mechanism: (1) altering proangiogenic BC secretome/TME and (2) inhibiting VEC growth. It may represent a therapeutic molecule to target breast tumor growth.

Zinc Aluminate-Based Composite Nanoparticles for Microwave Applications.

This paper reports the sol-gel preparation of ZnAl2O4 (ZA) and ZnAl2O4-TiO2 (ZAT) dielectric ceramic nanoparticles for fabricating prototype microstrip patch antennas. The prepared nanoparticles were polycrystalline in nature with their crystallite sizes of 9.4 and 11 nm, along with average grain diameters of 16 and 12 nm corresponding to samples ZA and ZAT. Dielectric properties were investigated using an LCR meter, which endorsed enhanced dielectric permittivity and decreased dielectric loss. Finally, prototype microstrip patch antennas named AZA and AZAT were fabricated using the prepared nanoparticles, and their performances were evaluated. Both antennas exhibited resonant peaks in the frequency range from 6.4 to 6.5 GHz. The antenna AZAT showed a return loss of -37.07 dB with a voltage standing wave ratio (VSWR) of 1.02 compared to the return loss of -19.42 dB, and a VSWR of 1.24 corresponds to AZA. The AZAT antenna's improved return loss can be regarded as the increased dielectric permittivity and reduced tangent loss of the ZAT sample. Furthermore, the ZAT antenna evidenced increased/decreased forwarded/reflected power decreased reflection coefficient and an optimal VSWR value compared to that of the AZA antenna.

Transcriptomic and Functional Evidence for Differential Effects of MCF-7 Breast Cancer Cell-Secretome on Vascular and Lymphatic Endothelial Cell Growth.

Vascular and lymphatic vessels drive breast cancer (BC) growth and metastasis. We assessed the cell growth (proliferation, migration, and capillary formation), gene-, and protein-expression profiles of Vascular Endothelial Cells (VECs) and Lymphatic Endothelial Cells (LECs) exposed to a conditioned medium (CM) from estrogen receptor-positive BC cells (MCF-7) in the presence or absence of Estradiol. We demonstrated that MCF-7-CM stimulated growth and capillary formation in VECs but inhibited LEC growth. Consistently, MCF-7-CM induced ERK1/2 and Akt phosphorylation in VECs and inhibited them in LECs. Gene expression analysis revealed that the LECs were overall (≈10-fold) more sensitive to MCF-7-CM exposure than VECs. Growth/angiogenesis and cell cycle pathways were upregulated in VECs but downregulated in LECs. An angiogenesis proteome array confirmed the upregulation of 23 pro-angiogenesis proteins in VECs. In LECs, the expression of genes related to ATP synthesis and the ATP content were reduced by MCF-7-CM, whereas MTHFD2 gene, involved in folate metabolism and immune evasion, was upregulated. The contrasting effect of MCF-7-CM on the growth of VECs and LECs was reversed by inhibiting the TGF-ß signaling pathway. The effect of MCF-7-CM on VEC growth was also reversed by inhibiting the VEGF signaling pathway. In conclusion, BC secretome may facilitate cancer cell survival and tumor growth by simultaneously promoting vascular angiogenesis and inhibiting lymphatic growth. The differential effects of BC secretome on LECs and VECs may be of pathophysiological relevance in BC.

Estradiol Inhibits Human Brain Vascular Pericyte Migration Activity: A Functional and Transcriptomic Analysis.

Stroke is the third leading cause of mortality in women and it kills twice as many women as breast cancer. A key role in the pathophysiology of stroke plays the disruption of the blood-brain barrier (BBB) within the neurovascular unit. While estrogen induces vascular protective actions, its influence on stroke remains unclear. Moreover, experiments assessing its impact on endothelial cells to induce barrier integrity are non-conclusive. Since pericytes play an active role in regulating BBB integrity and function, we hypothesize that estradiol may influence BBB by regulating their activity. In this study using human brain vascular pericytes (HBVPs) we investigated the impact of estradiol on key pericyte functions known to influence BBB integrity. HBVPs expressed estrogen receptors (ER-α, ER-ß and GPER) and treatment with estradiol (10 nM) inhibited basal cell migration but not proliferation. Since pericyte migration is a hallmark for BBB disruption following injury, infection and inflammation, we investigated the effects of estradiol on TNFα-induced PC migration. Importantly, estradiol prevented TNFα-induced pericyte migration and this effect was mimicked by PPT (ER-α agonist) and DPN (ER-ß agonist), but not by G1 (GPR30 agonist). The modulatory effects of estradiol were abrogated by MPP and PHTPP, selective ER-α and ER-ß antagonists, respectively, confirming the role of ER-α and ER-ß in mediating the anti-migratory actions of estrogen. To delineate the intracellular mechanisms mediating the inhibitory actions of estradiol on PC migration, we investigated the role of AKT and MAPK activation. While estradiol consistently reduced the TNFα-induced MAPK and Akt phosphorylation, only the inhibition of MAPK, but not Akt, significantly abrogated the migratory actions of TNFα. In transendothelial electrical resistance measurements, estradiol induced barrier function (TEER) in human brain microvascular endothelial cells co-cultured with pericytes, but not in HBMECs cultured alone. Importantly, transcriptomics analysis of genes modulated by estradiol in pericytes showed downregulation of genes known to increase cell migration and upregulation of genes known to inhibit cell migration. Taken together, our findings provide the first evidence that estradiol modulates pericyte activity and thereby improves endothelial integrity.

Proteomic Analysis of Estrogen-Mediated Enhancement of Mesenchymal Stem Cell-Induced Angiogenesis In Vivo.

Therapeutic use of mesenchymal stem cells (MSCs) for tissue repair has great potential. MSCs from multiple sources, including those derived from human umbilical matrix, namely Wharton's jelly, may serve as a resource for obtaining MSCs. However, low in vivo engraftment efficacy of MSCs remains a challenging limitation. To improve clinical outcomes using MSCs, an in-depth understanding of the mechanisms and factors involved in successful engraftment is required. We recently demonstrated that 17ß-estradiol (E2) improves MSCs in vitro proliferation, directed migration and engraftment in murine heart slices. Here, using a proteomics approach, we investigated the angiogenic potential of MSCs in vivo and the modulatory actions of E2 on mechanisms involved in tissue repair. Specifically, using a Matrigel® plug assay, we evaluated the effects of E2 on MSCs-induced angiogenesis in ovariectomized (OVX) mice. Moreover, using proteomics we investigated the potential pro-repair processes, pathways, and co-mechanisms possibly modified by the treatment of MSCs with E2. Using RT-qPCR, we evaluated mRNA expression of pro-angiogenic molecules, including endoglin, Tie-2, ANG, and VEGF. Hemoglobin levels, a marker for blood vessel formation, were increased in plugs treated with E2 + MSCs, suggesting increased capillary formation. This conclusion was confirmed by the histological analysis of capillary numbers in the Matrigel® plugs treated with E2 + MSC. The LC-MS screening of proteins obtained from the excised Matrigel® plugs revealed 71 proteins that were significantly altered following E2 exposure, 57 up-regulated proteins and 14 down-regulated proteins. A major result was the association of over 100 microRNA molecules (miRNAs) involved in cellular communication, vesicle transport, and metabolic and energy processes, and the high percentage of approximately 25% of genes involved in unknown biological processes. Together, these data provide evidence for increased angiogenesis by MSCs treated with the sex hormone E2. In conclusion, E2 treatment may increase the engraftment and repair potential of MSCs into tissue, and may promote MSC-induced angiogenesis after tissue injury.

Transcryptomic Analysis of Human Brain -Microvascular Endothelial Cell Driven Changes in -Vascular Pericytes.

Many pathological conditions of the brain are associated with structural abnormalities within the neurovascular system and linked to pericyte (PC) loss and/or dysfunction. Since crosstalk between endothelial cells (ECs) and PCs greatly impacts the function of the blood-brain barrier (BBB), effects of PCs on endothelial integrity and function have been investigated extensively. However, the impact of ECs on the function and activity of PCs remains largely unknown. Hence, using co-cultures of human brain vascular PCs with human cerebral microvascular ECs on opposite sides of porous Transwell inserts which facilitates direct EC-PC contact and improves EC barrier function, we analyzed EC-driven transcriptomic changes in PCs using microarrays and changes in cytokines/chemokines using proteome arrays. Gene expression analysis (GEA) in PCs co-cultured with ECs versus PCs cultured alone showed significant upregulation of 1'334 genes and downregulation of 964 genes. GEA in co-cultured PCs revealed increased expression of five prominent PC markers as well as soluble factors, such as transforming growth factor beta, fibroblast growth factor, angiopoietin 1, brain-derived neurotrophic factor, all of which are involved in EC-PC crosstalk and BBB induction. Pathway enrichment analysis of modulated genes showed a strong impact on many inflammatory and extracellular matrix (ECM) pathways including interferon and interleukin signaling, TGF-ß and interleukin-1 regulation of ECM, as well as on the mRNA processing pathway. Interestingly, while co-culture induced the mRNA expression of many chemokines and cytokines, including several CCL- and CXC-motif ligands and interleukins, we observed a decreased expression of the same inflammatory mediators on the protein level. Importantly, in PCs, ECs significantly induced interferon associated proteins (IFIT1, IFI44L, IF127, IFIT3, IFI6, IFI44) with anti-viral actions; downregulated prostaglandin E receptor 2 (prevent COX-2 mediated BBB damage); upregulated fibulin-3 and connective tissue growth factor essential for BBB integrity; and multiple ECMs (collagens and integrins) that inhibit cell migration. Our findings suggest that via direct contact, ECs prime PCs to induce molecules to promote BBB integrity and cell survival during infection and inflammatory insult. Taken together, we provide first evidence that interaction with ECs though porous membranes induces major changes in the transcriptomic and proteomic profile of PCs. ECs influence genes involved in diverse aspects of PC function including PC maturation, cell survival, anti-viral defense, blood flow regulation, immuno-modulation and ECM deposition.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved. This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens.

Autocrine/paracrine factors generated in response to 17ß-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE's; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (ß-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2',5'-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE's (genistein, 4-hydroxy-2',4',6'-trichlorobiphenyl, 4-hydroxy-2',4',6'-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/ß antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE's stimulate cAMP synthesis via GPER. Exposure of the FT to EE's and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.

Transcryptomic Analysis of Human Brain-Microvascular Endothelial Response to -Pericytes: Cell Orientation Defines Barrier Function.

Pericytes facilitate blood-brain barrier (BBB) integrity; however, the mechanisms involved remain unclear. Hence, using co-cultures of human cerebral microvascular endothelial cells (ECs) and vascular pericytes (PCs) in different spatial arrangements, as well as PC conditioned media, we investigated the impact of PC-EC orientation and PC-derived soluble factors on EC barrier function. We provide the first evidence that barrier-inducing properties of PCs require basolateral contact with ECs. Gene expression analysis (GEA) in ECs co-cultured with PCs versus ECs alone showed significant upregulation of 38 genes and downregulation of 122 genes. Pathway enrichment analysis of modulated genes showed significant regulation of several pathways, including transforming growth factor-ß and interleukin-1 regulated extracellular matrix, interferon and interleukin signaling, immune system signaling, receptor of advanced glycation end products (RAGE), and cytokine-cytokine receptor interaction. Transcriptomic analysis showed a reduction in molecules such as pro-inflammatory cytokines and chemokines, which are known to be induced during BBB disruption. Moreover, cytokine proteome array confirmed the downregulation of key pro-inflammatory cytokines and chemokines on the protein level. Other molecules which influence BBB and were favorably modulated upon EC-PC co-culture include IL-18 binding protein, kallikrein-3, CSF2 CSF3, CXCL10, CXCL11 (downregulated) and IL-1-R4; HGF, PDGF-AB/BB, PECAM, SERPIN E1 (upregulated). In conclusion, we provide the first evidence that (1) basolateral contact between ECs and PCs is essential for EC barrier function and integrity; (2) in ECs co-cultured with PCs, the profile of BBB disrupting pro-inflammatory molecules and cytokines/chemokines is downregulated; (3) PCs significantly modulate EC mechanisms known to improve barrier function, including TGF-ß regulated ECM pathway, anti-inflammatory cytokines, growth factors and matrix proteins. This human PC-EC co-culture may serve as a viable in vitro model for investigating BBB function and drug transport.

Synthesis and Characterization of Various Doped TiO2 Nanocrystals for Dye-Sensitized Solar Cells.

Few works are reported on solvothermal preparation of nanoparticles by utilizing acetone alone without a surfactant. This synthesis approach is found to be prominent for producing the mesoporous structure, which is crucial in improving the dye loading of the photoanode. In addition, doping of metal ions is advantageous in order to bring down the excitation energy, which is promising for boosting the performance of the doped oxides. This research aims to synthesize various kinds of doped-TiO2 nanocrystals to serve as photoanode materials in dye-sensitized solar cells (DSSCs). An X-ray diffraction study evidenced the existence of the crystalline phase in pure and doped-TiO2 nanocrystals. Rietveld refinement study showed the mixed phases of crystalline TiO2 in the CrT, CuNT, and ST as compared to a single anatase phase in the samples PT, AgT, BT, CoT, FeT, SnT, ZT, VT, and ZMT. The absorption spectroscopy analysis demonstrated the reduced optical band gap from 3.10 to 2.79 eV. Scanning electron microscopy investigation endorsed the formation of TiO2 mesoporous microspheres with a mean diameter ranging from 200 to 331 nm along with a nanocrystal diameter ranging from 10 to 20 nm. Doping with the different dopants enhanced the conversion efficiency of DSSCs from 1.31 to ∼6%. Furthermore, we have performed the electrochemical impedance spectroscopy of DSSCs, and the findings are presented.

Exploration of Diosmin to Control Diabetes and Its Complications-an In Vitro and In Silico Approach.

BACKGROUND: Diosmin is a flavonoid obtained from the citrus fruits of the plants. Diosmin has blood lipid lowering activities, antioxidant activity, enhances venous tone and microcirculation, protects capillaries, mainly by reducing systemic oxidative stress. OBJECTIVE: The present study demonstrates the potential of Diosmin against the enzymes aldose reductase, α-glucosidase, and α-amylase involved in diabetes and its complications by in vitro evaluation and reverse molecular docking studies. METHODS: The assay of aldose reductase was performed by using NADPH as starting material and DL-Glyceraldehyde as a substrate. DNS method was used for alpha amylase inhibition and in alpha glucosidase inhibitory activity p-nitrophenyl glucopyranoside (pNPG) was used as substrate. The reverse molecular docking studies was performed by using Molegro software (MVD) with grid resolution of 30 Å. RESULTS: Diosmin shows potent inhibitory effect against aldose reductase (IC50:333.88±0.04 µg/mL), α-glucosidase (IC50:410.3±0.01 µg/mL) and α-amylase (IC50: 404.22±0.02 µg/mL) respectively. The standard drugs shows moderate inhibitory activity for enzymes. The MolDock Score of Diosmin was -224.127 against aldose reductase, -168.17 against α-glucosidase and - 176.013 against α-amylase respectively, which was much higher than standard drugs. CONCLUSION: From the result it was concluded that diosmin was a potentially inhibitor of aldose reductase, alpha amylase and alpha glucosidase enzymes then the standard drugs and it will be helpful in the management of diabetes and its complications. This will also be benevolent to decrease the socio economical burden on the middle class family of the society.

Molecular Docking Studies of Bioactive Nicotiflorin against 6W63 Novel Coronavirus 2019 (COVID-19).

BACKGROUND: COVID-19 which is known as the novel coronavirus was reported in December 2019 in Wuhan city, China and many people have been contaminated by environmental contamination and transmission from one human to another until now. OBJECTIVE: The objective of the present work is to establish the inhibitory potential of nicotiflorin, a Kaempferol 3-O-rutinoside flavonoid, against the deadly coronavirus (COVID-19) 6W63 (main protease 3Clpro protein), using molecular docking approach. METHODS: The Molegro Virtual Docker software (MVD) with a 30 Å grid resolution was used. The structure was drawn by Chem 3D software and energy minimization was done by the MM2 force field. The protein 6W63 was downloaded from the protein data bank. Molegro modeller was used for score calculations. RESULT: The molecular docking studies were carried out on nicotiflorin and standard inhibitor X77, where standard inhibitor was observed in a co-crystallized state with main protease 3Clpro protein 6W63. The MolDock score, Rerank Sore, and H Bond score of nicotiflorin and standard inhibitor X77 were observed as -173.058, -127.302, -21.9398 and -156.913,-121.296,-5.7369, respectively. CONCLUSION: Molecular docking studies have confirmed that the affinity of flavonoid nicotiflorin with the amino acids of the viral protein 6W63 was relatively more than the standard X77. For the effective treatment of novel coronavirus COVID-19, the effectiveness of the identified flavonoid nicotiflorin can further be evaluated for safety and efficacy parameters at both preclinical and clinical stages.

Computation screening of narcissoside a glycosyloxyflavone for potential novel coronavirus 2019 (COVID-19) inhibitor.

Background: The present study demonstrates the potential of flavanoid narcissoside against the novel corona virus (COVID-19) complications using molecular docking studies. Methods: The computation molecular docking screening was performed using Molegro Virtual Docker software (MVD) with grid resolution of 30 Å. Protein of COVID 19 virus was taken from protein data bank. Results: The standard inhibitor X77 (N-(4-tert-butylphenyl)-N-[(1R)-2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl]-1H-imidazole-4-carboxamide) identified from the protein inhibitor complex 6W63 from protein data bank was docked with COVID 19 protein 6W63 which showed MolDock score of -156.913, rerank Sore -121.296 and H Bond -5.7369, while the flavanoid narcissoside had showed MolDock score -180.739, Rerank Sore -137.092 and H Bond -18.6771. The narcissoside showed potent inhibitory effect which is greater than standard X77. The result showed that narcissoside have high affinity towards 6W63 as it showed thirteen hydrogen bonds with nine amino acids (Arg 188, Glu 166, His 164, Cys 145 (2 bonds), Asn 14 (2 bonds), Cys 44 (2 bonds), His 41 (2 bonds), Gln 192, Thr 190) while X777 showed four hydrogen bonds with amino acids (Gly 143, Cys 145, Glu 166, Ser 144). Conclusion: From computation approach it was concluded that narcissoside is a potent inhibitor of viral COVID 19 protein 6W63. The narcissoside have high affinity and inhibition potential than standard inhibitor X77 (N-(4-tert-butylphenyl)-N-[(1R)-2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl]-1H-imidazole-4-carboxamide). The narcissoside predicted as more potent inhibitor which can be further optimize, pharmacologically and clinically evaluated for the treatment of novel coronavirus COVID-19.

Adenosine, Via A2B Receptors, Inhibits Human (P-SMC) Progenitor Smooth Muscle Cell Growth.

c-Kit+ progenitor smooth muscle cells (P-SMCs) can develop into SMCs that contribute to injury-induced neointimal thickening. Here, we investigated whether adenosine reduces P-SMC migration and proliferation and whether this contributes to adenosine's inhibitory actions on neointima formation. In human P-SMCs, 2-chloroadenosine (stable adenosine analogue) and BAY60-6583 (A2B agonist) inhibited P-SMC proliferation and migration. Likewise, increasing endogenous adenosine by blocking adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl) adenine (inhibits adenosine deaminase) and 5-iodotubercidin (inhibits adenosine kinase) attenuated P-SMC proliferation and migration. Neither N6-cyclopentyladenosine (A1 agonist), CGS21680 (A2A agonist), nor N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (A3 agonist) affected P-SMC proliferation or migration. 2-Chloroadenosine increased cyclic AMP, reduced Akt phosphorylation (activates cyclin D expression), and reduced levels of cyclin D1 (promotes cell-cycle progression). Moreover, 2-chloroadenosine inhibited expression of Skp2 (promotes proteolysis of p27Kip1) and upregulated levels of p27Kip1 (negative cell-cycle regulator). A2B receptor knockdown prevented the effects of 2-chloroadenosine on cyclic AMP production and P-SMC proliferation and migration. Likewise, inhibition of adenylyl cyclase and protein kinase A rescued P-SMCs from the inhibitory effects of 2-chloroadenosine. The inhibitory effects of adenosine were similar in male and female P-SMCs. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 µmol/L for 7 days) reduced neointimal hyperplasia by 64.5% (P<0.05; intima/media ratio: control, 1.4±0.02; treated, 0.53±0.012) and reduced neointimal c-Kit+ cells. Adenosine inhibits P-SMC migration and proliferation via the A2B receptor/cyclic AMP/protein kinase A axis, which reduces cyclin D1 expression and activity via inhibiting Akt phosphorylation and Skp2 expression and upregulating p27kip1 levels. Adenosine attenuates neointima formation in part by inhibiting infiltration and proliferation of c-Kit+ P-SMCs.

Mechanism of 17ß-estradiol stimulated integration of human mesenchymal stem cells in heart tissue.

Scarcity of gender specific donor hearts highlights the importance of mesenchymal stem cells (MSCs) as a therapeutic tool for heart repair. However, inefficient incorporation, retention, and activity of MSCs in cardiac tissue remain an obstacle. Since surges in follicular estradiol (E2; µmolar-range) trigger tissue remodeling (e.g. ovulation) and E2 exerts beneficial actions on the cardiovascular system, we hypothesized that E2 may promote/improve MSC-mediated cardiac repair processes. Using Wharton's jelly (WJ)-derived MSCs we assessed the effects of E2 on MSC proliferation, directed migration, and engraftment in murine heart slices (using xCELLigence real-time cell-impedance system, DNA quantification, and microscopy) and on MSC-induced angiogenesis in vivo (matrigel plug assay). Protein expression was assessed by Western blotting, ELISA/Luminex, and proteomic analysis; whereas mRNA expression was assessed by qRT-PCR. MSCs expressed estrogen receptors (ERs) -alpha and -beta. E2 promoted MSC proliferation and up-regulated mRNA and protein expression of ER-alpha, ER-beta, extracellular matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase (MMP) -9, yet down-regulated MMP-2 expression. Moreover, E2 up-regulated expression of vascular endothelial growth factor (VEGF)-A, VEGFR-2, vascular cell adhesion protein-1 (VCAM-1), and angiogenin (ANG) and stimulated nitric oxide (NO) production via ER. Proteomic analysis of MSCs showed that E2 up-regulated 47 proteins, down-regulated 7 proteins, and increased the expression of key biochemical components/pathways involved in tissue repair. In MSCs co-cultured with murine heart-slices, E2 significantly induced MSC migration in an ER-alpha-dependent fashion and significantly increased the secretion of MMP-2, MMP-9, ANG, and VEGF. In an in vivo matrigel assay, E2-treated MSCs increased angiogenesis and hemoglobin content. In conclusion, E2-treatment increases the incorporation of MSCs in heart slices and promotes MSC-induced angiogenesis. These beneficial effects are mediated via increases in molecules/pathways involved in tissue remodeling and angiogenesis. We speculate that E2 may enhance MSC ability to repair/regenerate cardiac tissue.

Dihydrotestosterone induces pro-angiogenic factors and assists homing of MSC into the cardiac tissue.

The use of mesenchymal stem cells (MSC) as a therapeutic tool in cardiovascular diseases is promising. Since androgens exert some beneficial actions on the cardiovascular system, we tested our hypothesis that this hormone could promote MSC-mediated repair processes, also. Cultured MSCs isolated from Wharton's jelly were exposed to 30 nM dihydrotestosterone (DHT) for 1 or 4 days and the effects of the hormone on their growth/migration/adhesion and the underlying mechanisms were assessed. Results were obtained by real-time cell impedance measurements, and DNA quantification showed that DHT increased MSC proliferation by ~30%. As determined by xCELLigence system, DHT augmented (~2 folds) the migration of MSC toward cardiac tissue slices (at 12 h), and this effect was blocked by flutamide, an androgen receptor (AR) antagonist. Exposure of cells to DHT, upregulated the gene and protein expression of AR, EMMPRIN and MMP-9 and downregulated the expression of MMP-2 DHT significantly induced the release of nitric oxide by MSC (≥2-fold) and flutamide blocked this effect. When MSCs were co-cultured with cardiac slices, immunohistochemical analysis and qRT-PCR showed that the integration of DHT-stimulated MSC was significantly higher than that of in controls. In conclusion, our findings provide the first evidence that DHT promotes MSC growth, migration and integration into the cardiac slices. The modulating effects of DHT were associated with upregulation of ARs and of key molecules known to promote tissue remodeling and angiogenesis. Our findings suggest that priming of MSC with DHT may potentially increase their capability to regenerate cardiac tissue; in vivo studies are needed to confirm our in vitro findings.