14K prolactin derived 14-mer antiangiogenic peptide targets bradykinin-/nitric oxide-cGMP-dependent angiogenesis.

Over the past few decades, VEGF-targeted antiangiogenic therapy for cancers has gained increasing attention. Nevertheless, there are still several limitations such as the potential resistance mechanisms arising in cancer cells against these therapies and their potential adverse effects. These limitations highlight the need for novel anti-angiogenesis molecules and better understanding of the mechanisms of tumor angiogenesis. In the present study, we investigated the antiangiogenic properties of a novel 14-mer antiangiogenic peptide (14-MAP) derived from N-terminal 14 kDa buffalo prolactin and characterized its mode of action. 14-MAP at the picomolar concentration inhibited VEGF- and bradykinin (an autacoid peptide expressed in vascular tissues in pathophysiology, BK)-stimulated endothelial nitric oxide (eNO) production, cell migration, and proliferation in endothelial cells and vessel development in the chick embryo. Although this peptide inhibited both VEGF- and BK-dependent angiogenic processes, its action was more pronounced in the latter. Moreover, the interference of 14-MAP with the eNO synthase (eNOS)-cyclic GMP pathway was also identified. A combination of a low dose of Avastin, a widely used drug targeting VEGF-dependent angiogenesis, and 14-MAP significantly reduced tumor size in an in vivo model of human colon cancer. Taken together, our results suggest that 14-MAP, a BK- and eNOS-dependent antiangiogenic peptide, might be useful for overcoming the limitation of VEGF-targeted antiangiogenic therapy in cancer patients. However, further studies will be required to further characterize its mode of action and therapeutic potential.

Effect of the Addition of Inorganic Fillers on the Properties of Degradable Polymeric Blends for Bone Tissue Engineering.

Bone tissue exhibits self-healing properties; however, not all defects can be repaired without surgical intervention. Bone tissue engineering offers artificial scaffolds, which can act as a temporary matrix for bone regeneration. The aim of this study was to manufacture scaffolds made of poly(lactic acid), poly(ε-caprolactone), poly(propylene fumarate), and poly(ethylene glycol) modified with bioglass, beta tricalcium phosphate (TCP), and/or wollastonite (W) particles. The scaffolds were fabricated using a gel-casting method and observed with optical and scanning electron microscopes. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR), differential scanning calorimetry (DSC), thermogravimetry (TG), wettability, and degradation tests were conducted. The highest content of TCP without W in the composition caused the highest hydrophilicity (water contact angle of 61.9 ± 6.3°), the fastest degradation rate (7% mass loss within 28 days), moderate ability to precipitate CaP after incubation in PBS, and no cytotoxicity for L929 cells. The highest content of W without TCP caused the highest hydrophobicity (water contact angle of 83.4 ± 1.7°), the lowest thermal stability, slower degradation (3% mass loss within 28 days), and did not evoke CaP precipitation. Moreover, some signs of cytotoxicity on day 1 were observed. The samples with both TCP and W showed moderate properties and the best cytocompatibility on day 4. Interestingly, they were covered with typical cauliflower-like hydroxyapatite deposits after incubation in phosphate-buffered saline (PBS), which might be a sign of their excellent bioactivity.

Characterization and In Vitro Evaluation of Porous Polymer-Blended Scaffolds Functionalized with Tricalcium Phosphate.

Bone tissue is one of the most transplanted tissues. The ageing population and bone diseases are the main causes of the growing need for novel treatments offered by bone tissue engineering. Three-dimensional (3D) scaffolds, as artificial structures that fulfil certain characteristics, can be used as a temporary matrix for bone regeneration. In this study, we aimed to fabricate 3D porous polymer scaffolds functionalized with tricalcium phosphate (TCP) particles for applications in bone tissue regeneration. Different combinations of poly(lactic acid) (PLA), poly(ethylene glycol) (PEG with molecular weight of 600 or 2000 Da) and poly(ε-caprolactone) (PCL) with TCP were blended by a gel-casting method combined with rapid heating. Porous composite scaffolds with pore sizes from 100 to 1500 µm were obtained. ATR-FTIR, DSC, and wettability tests were performed to study scaffold composition, thermal properties, and hydrophilicity, respectively. The samples were observed with the use of optical and scanning electron microscopes. The addition of PCL to PLA increased the hydrophobicity of the composite scaffolds and reduced their susceptibility to degradation, whereas the addition of PEG increased the hydrophilicity and degradation rates but concomitantly resulted in enhanced creation of rounded mineral deposits. The scaffolds were not cytotoxic according to an indirect test in L929 fibroblasts, and they supported adhesion and growth of MG-63 cells when cultured in direct contact.

Green Synthesis of Silver Nanoparticles Using the Leaf Extract of the Medicinal Plant, Uvaria narum and Its Antibacterial, Antiangiogenic, Anticancer and Catalytic Properties.

Silver nanoparticles (AgNPs) made by green synthesis offer a variety of biochemical properties and are an excellent alternative to traditional medications due to their low cost. In the current study, we synthesised AgNPs from the leaf extract of the medicinal plant Uvaria narum, commonly called narumpanal. The nanoparticles were characterised by ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM analysis showed AgNPs are highly crystalline and spherical with an average diameter of 7.13 nm. The outstanding catalytic activity of AgNPs was demonstrated by employing the reduction of 4-nitrophenol to 4-aminophenol. The AgNPs showed antiangiogenic activity in the chick chorioallantoic membrane (CAM) assay. AgNPs demonstrated anticancer activity against Dalton's lymphoma ascites cells (DLA cells) in trypan blue assay and cytotoxicity against three fish cell lines: Oreochromis niloticus liver (onlL; National Repository of Fish Cell Lines, India (NRFC) Accession number-NRFC052) cells, Cyprinus carpio koi fin (CCKF; NRFC Accession number-NRFC007) cells and Cyprinus carpio gill (CyCKG; NRFC Accession number-NRFC064). Furthermore, the AgNPs demonstrated their ability to inhibit pathogenic microorganisms, Staphylococcus aureus, and Escherichia coli. The results from the study displayed green synthesised AgNPs exhibit antiangiogenic activity, cytotoxicity, antimicrobial and catalytic properties, which are crucial characteristics of a molecule with excellent clinical applications.

RGS11-CaMKII complex mediated redox control attenuates chemotherapy-induced cardiac fibrosis.

Dose limiting cardiotoxicity remains a major limiting factor in the clinical use of several cancer chemotherapeutics including anthracyclines and the antimetabolite 5-fluorouracil (5-FU). Prior work has demonstrated that chemotherapeutics increase expression of R7 family regulator of G protein signaling (RGS) protein-binding partner Gß5, which drives myocyte cytotoxicity. However, though several R7 family members are expressed in heart, the exact role of each protein in chemotherapy driven heart damage remains unclear. Here, we demonstrate that RGS11, downregulated in the human heart following chemotherapy exposure, possesses potent anti-apoptotic actions, in direct opposition to the actions of fellow R7 family member RGS6. RGS11 forms a direct complex with the apoptotic kinase CaMKII and stress responsive transcription factor ATF3 and acts to counterbalance the ability of CaMKII and ATF3 to trigger oxidative stress, mitochondrial dysfunction, cell death, and release of the cardiokine neuregulin-1 (NRG1), which mediates pathological intercommunication between myocytes and endothelial cells. Doxorubicin triggers RGS11 depletion in the murine myocardium, and cardiac-specific OE of RGS11 decreases doxorubicin-induced fibrosis, myocyte hypertrophy, apoptosis, oxidative stress, and cell loss and aids in the maintenance of left ventricular function. Conversely, RGS11 knockdown in heart promotes cardiac fibrosis associated with CaMKII activation and ATF3/NRG1 induction. Indeed, inhibition of CaMKII largely prevents the fibrotic remodeling resulting from cardiac RGS11 depletion underscoring the functional importance of the RGS11-CaMKII interaction in the pathogenesis of cardiac fibrosis. These data describe an entirely new role for RGS11 in heart and identify RGS11 as a potential new target for amelioration of chemotherapy-induced cardiotoxicity.

Nitric oxide donor 8-bromo-cGMP suppresses cholesterol depleted RBCs band 3 dysfunction.

Red blood cells (RBCs) carry large cholesterol fractions and imbalance in them leads to several vascular complications. RBCs band 3 protein plays an important role in maintaining membrane integrity and there are many reports on cholesterol and band 3 protein interaction. Yet, RBCs band 3 protein role in regulating cholesterol homeostasis needs to be investigated. In this study, we induced cholesterol-depletion and band 3 inhibition in RBCs; both of which cause stress by decreasing band 3 channel activity with an increase in RBCs adhesion to endothelial cells (EC) by elevating band 3 phosphorylation (Tyr21), methemoglobin level and decreasing nitric oxide level. We hypothesized that nitric oxide (NO), a prominent determinant for RBC structural stability, would protect RBCs from stressors. To estimate this, we used three NO donors (SpNO, Sildenafil citrate and 8-Bromo-cGMP) and found that all 3 NO donors were able to recover, with 8-Bromo-cGMP being the most effective as it not only increased band 3 channel activity but also decreased RBC-EC adhesiveness and methemoglobin level in both stressors. Whereas NO donor's treatment did not display an ameliorative impact when both stresses were combined. Overall, these findings may shed light on the role of 8-bromo-cGMP in regulating RBC cholesterol homeostasis by maintaining band 3 function. Further studies in this direction might help identify targets for the therapeutic use of NO donors in the treatment of blood disorders.

COVID-19 and dys-regulation of pulmonary endothelium: implications for vascular remodeling.

Coronavirus disease-2019 (COVID-19), the disease caused by severe acute respiratory syndrome-coronavirus-2, has claimed more than 4.4 million lives worldwide (as of 20 August 2021). Severe cases of the disease often result in respiratory distress due to cytokine storm, and mechanical ventilation is required. Although, the lungs are the primary organs affected by the disease, more evidence on damage to the heart, kidney, and liver is emerging. A common link in these connections is the cardiovascular network. Inner lining of the blood vessels, called endothelium, is formed by a single layer of endothelial cells. Several clinical manifestations involving the endothelium have been reported, such as its activation via immunomodulation, endotheliitis, thrombosis, vasoconstriction, and distinct intussusceptive angiogenesis (IA), a unique and rapid process of blood-vessel formation by splitting a vessel into two lumens. In fact, the virus directly infects the endothelium via TMPRSS2 spike glycoprotein priming to facilitate ACE-2-mediated viral entry. Recent studies have indicated a significant increase in remodeling of the pulmonary vascular bed via intussusception in patients with COVID-19. However, the lack of circulatory biomarkers for IA limits its detection in COVID-19 pathogenesis. In this review, we describe the implications of angiogenesis in COVID-19, unique features of the pulmonary vascular bed and its remodeling, and a rapid and non-invasive assessment of IA to overcome the technical limitations in patients with COVID-19.

Temporal dynamics of nitric oxide wave in early vasculogenesis.

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10-HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

Inhibition of hypoxia-induced Mucin 1 alters the proteomic composition of human osteoblast-produced extracellular matrix, leading to reduced osteogenic and angiogenic potential.

The bone microenvironment is one of the most hypoxic regions of the human body and in experimental models; hypoxia inhibits osteogenic differentiation of mesenchymal stromal cells (MSCs). Our previous work revealed that Mucin 1 (MUC1) was dynamically expressed during osteogenic differentiation of human MSCs and upregulated by hypoxia. Upon stimulation, its C-terminus (MUC1-CT) is proteolytically cleaved, translocases to the nucleus, and binds to promoters of target genes. Therefore, we assessed the MUC1-mediated effect of hypoxia on the proteomic composition of human osteoblast-derived extracellular matrices (ECMs) and characterized their osteogenic and angiogenic potentials in the produced ECMs. We generated ECMs from osteogenically differentiated human MSC cultured in vitro under 20% or 2% oxygen with or without GO-201, a MUC1-CT inhibitor. Hypoxia upregulated MUC1, vascular endothelial growth factor, and connective tissue growth factor independent of MUC1 inhibition, whereas GO-201 stabilized hypoxia-inducible factor 1-alpha. Hypoxia and/or MUC1-CT inhibition reduced osteogenic differentiation of human MSC by AMP-activated protein kinase/mTORC1/S6K pathway and dampened their matrix mineralization. Hypoxia modulated ECMs by transforming growth factor-beta/Smad and phosphorylation of NFκB and upregulated COL1A1, COL5A1, and COL5A3. The ECMs of hypoxic osteoblasts reduced MSC proliferation and accelerated their osteogenic differentiation, whereas MUC1-CT-inhibited ECMs counteracted these effects. In addition, ECMs generated under MUC1-CT inhibition reduced the angiogenic potential independent of oxygen concentration. We claim here that MUC1 is critical for hypoxia-mediated changes during osteoblastogenesis, which not only alters the proteomic landscape of the ECM but thereby also modulates its osteogenic and angiogenic potentials.

Cholecalciferol and metformin protect against lipopolysaccharide-induced endothelial dysfunction and senescence by modulating sirtuin-1 and protein arginine methyltransferase-1.

Endothelial cell activation through nuclear factor-kappa-B (NFkB) and mitogen-activated protein kinases leads to increased biosynthesis of pro-inflammatory mediators, cellular injury and vascular inflammation under lipopolysaccharide (LPS) exposure. Recent studies report that LPS up-regulated global methyltransferase activity. In this study, we observed that a combination treatment with metformin (MET) and cholecalciferol (VD) blocked the LPS-induced S-adenosylmethionine (SAM)-dependent methyltransferase (SDM) activity in Eahy926 cells. We found that LPS challenge (i) increased arginine methylation through up-regulated protein arginine methyltransferase-1 (PRMT1) mRNA, intracellular concentrations of asymmetric dimethylarginine (ADMA) and homocysteine (HCY); (ii) up-regulated cell senescence through mitigated sirtuin-1 (SIRT1) mRNA, nicotinamide adenine dinucleotide (NAD+) concentration, telomerase activity and total antioxidant capacity; and (iii) lead to endothelial dysfunction through compromised nitric oxide (NOx) production. However, these LPS-mediated cellular events in Eahy926 cells were restored by the synergistic effect of MET and VD. Taken together, this study identified that the dual compound effect inhibits LPS-induced protein arginine methylation, endothelial senescence and dysfunction through the components of epigenetic machinery, SIRT1 and PRMT1, which is a previously unidentified function of the test compounds. In silico results identified the presence of vitamin D response element (VDRE) sequence on PRMT1 suggesting that VDR could regulate PRMT1 gene expression. Further characterization of the cellular events associated with the dual compound challenge, using gene silencing approach or adenoviral constructs for SIRT1 and/or PRMT1 under inflammatory stress, could identify therapeutic strategies to address the endothelial consequences in vascular inflammation-mediated atherosclerosis.

Nitric oxide mitigates thalidomide-induced abnormalities during germination and development of fennel seeds.

Thalidomide causes teratogenic effects in several animal species and in humans. Accordingly, the World Health Organization banned thalidomide when mothers who took thalidomide during pregnancy delivered abnormal fetuses. After four decades, thalidomide underwent drug "re-purposing" since its antiangiogenic and immunomodulatory effects were therapeutic for multiple myeloma. There are no reports of thalidomide's effects on prokaryotes, but it showed teratogenic effects in Arabidopsis thaliana, an ancestor of the plant kingdom. This proof of concept study clearly shows that thalidomide caused a significant and reproducible decrease in germination rate, nitric oxide (NO) production, and chlorophyll content of fennel plantlets. Thalidomide also induced the formation of abnormal fennel plantlets with stunting, wrinkling, and curling of fennel shoots and leaves. Notably, quantitative analysis showed that thalidomide caused a 50% increase in the formation of abnormal fennel plantlets and that these negative effects of thalidomide showed a 2.50- to 4-fold decrease when fennel seeds were co-incubated with an NO donor (Spermine NoNoate) or a stable cGMP analog 8-bromo Guanosine 3',5'-cyclic monophosphate (8-Bromo-cGMP). This study is important because it confirms that thalidomide's negative effects on fennel seed germination and growth are mediated by attenuation of NO and disruption of NO signaling. This reproducible model of thalidomide-induced, NO-dependent damage in a plant system can be used to further investigate the molecular mechanisms of thalidomide action in plants. Importantly, this study establishes a link between the evolution of development of higher plants and mammals.

Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death.

The pathophysiological mechanism(s) driving non-alcoholic fatty liver disease, the most prevalent chronic liver disease globally, have yet to be fully elucidated. Here, we identify regulator of G protein signaling 6 (RGS6), up-regulated in the livers of NAFLD patients, as a critical mediator of hepatic steatosis, fibrosis, inflammation, and cell death. Human patients with high hepatic RGS6 expression exhibited a corresponding high inflammatory burden, pronounced insulin resistance, and poor liver function. In mice, liver-specific RGS6 knockdown largely ameliorated high fat diet (HFD)-driven oxidative stress, fibrotic remodeling, inflammation, lipid deposition and cell death. RGS6 depletion allowed for maintenance of mitochondrial integrity restoring redox balance, improving fatty acid oxidation, and preventing loss of insulin receptor sensitivity in hepatocytes. RGS6 is both induced by ROS and increases ROS generation acting as a key amplification node to exacerbate oxidative stress. In liver, RGS6 forms a direct complex with ATM kinase supported by key aspartate residues in the RGS domain and is both necessary and sufficient to drive hyperlipidemia-dependent ATM phosphorylation. pATM and markers of DNA damage (γH2AX) were also elevated in livers from NAFLD patients particularly in samples with high RGS6 protein content. Unsurprisingly, RGS6 knockdown prevented ATM phosphorylation in livers from HFD-fed mice. Further, RGS6 mutants lacking the capacity for ATM binding fail to facilitate palmitic acid-dependent hepatocyte apoptosis underscoring the importance of the RGS6-ATM complex in hyperlipidemia-dependent cell death. Inhibition of RGS6, then, may provide a viable means to prevent or reverse liver damage by mitigating oxidative liver damage.

G protein ß5-ATM complexes drive acetaminophen-induced hepatotoxicity.

Excessive ingestion of the common analgesic acetaminophen (APAP) leads to severe hepatotoxicity. Here we identify G protein ß5 (Gß5), elevated in livers from APAP overdose patients, as a critical regulator of cell death pathways and autophagic signaling in APAP-exposed liver. Liver-specific knockdown of Gß5 in mice protected the liver from APAP-dependent fibrosis, cell loss, oxidative stress, and inflammation following either acute or chronic APAP administration. Conversely, overexpression of Gß5 in liver was sufficient to drive hepatocyte dysfunction and loss. In hepatocytes, Gß5 depletion ameliorated mitochondrial dysfunction, allowed for maintenance of ATP generation and mitigated APAP-induced cell death. Further, Gß5 knockdown also reversed impacts of APAP on kinase cascades (e.g. ATM/AMPK) signaling to mammalian target of rapamycin (mTOR), a master regulator of autophagy and, as a result, interrupted autophagic flux. Though canonically relegated to nuclear DNA repair pathways, ATM also functions in the cytoplasm to control cell death and autophagy. Indeed, we now show that Gß5 forms a direct, stable complex with the FAT domain of ATM, important for autophosphorylation-dependent kinase activation. These data provide a viable explanation for these novel, G protein-independent actions of Gß5 in liver. Thus, Gß5 sits at a critical nexus in multiple pathological sequelae driving APAP-dependent liver damage.

Repurposing of thalidomide and its derivatives for the treatment of SARS-coV-2 infections: Hints on molecular action.

AIMS: The SARS-coV-2 pandemic continues to cause an unprecedented global destabilization requiring urgent attention towards drug and vaccine development. Thalidomide, a drug with known anti-inflammatory and immunomodulatory effects has been indicated to be effective in treating a SARS-coV-2 pneumonia patient. Here, we study the possible mechanisms through which thalidomide might affect coronavirus disease-19 (COVID-19). METHODS: The present study explores the possibility of repurposing thalidomide for the treatment of SARS-coV-2 pneumonia by reanalysing transcriptomes of SARS-coV-2 infected tissues with thalidomide and lenalidomide induced transcriptomic changes in transformed lung and haematopoietic models as procured from databases, and further comparing them with the transcriptome of primary endothelial cells. RESULTS: Thalidomide and lenalidomide exhibited pleiotropic effects affecting a range of biological processes including inflammation, immune response, angiogenesis, MAPK signalling, NOD-like receptor signalling, Toll-like receptor signalling, leucocyte differentiation and innate immunity, the processes that are aberrantly regulated in severe COVID-19 patients. CONCLUSION: The present study indicates thalidomide analogues as a better fit for treating severe cases of novel viral infections, healing the damaged network by compensating the impairment caused by the COVID-19.

Ectopic release of nitric oxide modulates the onset of cardiac development in avian model.

Heart development is one of the earliest developmental events, and its pumping action is directly linked to the intensity of development of other organs. Heart contractions mediate the circulation of the nutrients and signalling molecules to the focal points of developing embryos. In the present study, we used in vivo, ex vivo, in vitro, and in silico methods for chick embryo model to characterize and identify molecular targets under the influence of ectopic nitric oxide in reference to cardiogenesis. Spermine NONOate (SpNO) treatment of 10 µM increased the percentage of chick embryos having beating heart at 40th h of incubation by 2.2-fold (p < 0.001). In an ex vivo chick embryo culture, SpNO increased the percentage of embryos having beats by 1.56-fold (p < 0.05) compared with control after 2 h of treatment. Total body weight of SpNO-treated chick embryos at the Hamburger and Hamilton (HH) stage 29 was increased by 1.22-fold (p < 0.005). Cardiac field potential (FP) recordings of chick embryo at HH29 showed 2.5-fold (p < 0.001) increased in the amplitude, 3.2-fold (p < 0.001) increased in frequency of SpNO-treated embryos over that of the control group, whereas FP duration was unaffected. In cultured cardiac progenitors cells (CPCs), SpNO treatment decreased apoptosis and cell death by twofold (p < 0.001) and 1.7-fold (p < 0.001), respectively. Transcriptome analysis of chick embryonic heart isolated from HH15 stage pre-treated with SpNO at HH8 stage showed upregulation of genes involved in heart morphogenesis, heart contraction, cardiac cell development, calcium signalling, structure, and development whereas downregulated genes were enriched under the terms extracellular matrix, wnt pathway, and BMP pathway. The key upstream molecules predicted to be activated were p38 MAPK, MEF2C, TBX5, and GATA4 while KDM5α, DNMT3A, and HNF1α were predicted to be inhibited. This study suggests that the ectopic nitric oxide modulates the onset of cardiac development.

Interactome of miRNAs and transcriptome of human umbilical cord endothelial cells exposed to short-term simulated microgravity.

Adaptation of humans in low gravity conditions is a matter of utmost importance when efforts are on to a gigantic leap in human space expeditions for tourism and formation of space colonies. In this connection, cardiovascular adaptation in low gravity is a critical component of human space exploration. Deep high-throughput sequencing approach allowed us to analyze the miRNA and mRNA expression profiles in human umbilical cord vein endothelial cells (HUVEC), cultured under gravity (G), and stimulated microgravity (MG) achieved with a clinostat. The present study identified totally 1870 miRNAs differentially expressed in HUVEC under MG condition when compared to the cells subjected to unitary G conditions. The functional association of identified miRNAs targeting specific mRNAs revealed that miRNAs, hsa-mir-496, hsa-mir-151a, hsa-miR-296-3p, hsa-mir-148a, hsa-miR-365b-5p, hsa-miR-3687, hsa-mir-454, hsa-miR-155-5p, and hsa-miR-145-5p differentially regulated the genes involved in cell adhesion, angiogenesis, cell cycle, JAK-STAT signaling, MAPK signaling, nitric oxide signaling, VEGF signaling, and wound healing pathways. Further, the q-PCR based experimental studies of upregulated and downregulated miRNA and mRNAs demonstrate that the above reported miRNAs influence the cell proliferation and vascular functions of the HUVEC in MG conditions effectively. Consensus on the interactome results indicates restricted fluctuations in the transcriptome of the HUVEC exposed to short-term MG that could lead to higher levels of endothelial functions like angiogenesis and vascular patterning.

Attenuation of cadmium-induced vascular toxicity by pro-angiogenic nanorods.

Cadmium (Cd) is a common heavy metal that causes major environmental pollution with adverse effects on human health and well-being. Exposure to Cd is known to exhibit detrimental consequences on all the vital organ systems of the body, especially the vascular system. Certain approaches using anti-oxidants and chelating agents have been demonstrated previously to mitigate Cd-induced toxicity. However, these approaches are associated with their own limitations. In this context, there is a critical need for the development of alternative treatment strategies to address the conditions associated with Cd-poisoning. One such novel approach is the application of nanomedicine which is well-known to resolve several health complications by improving disease therapy. Recently, our group demonstrated the role of europium hydroxide nanorods (EHN) in promoting vascular growth using in vitro and in vivo assay systems. Therefore, in the present study, we have evaluated the effect of EHN on health of endothelial cells (EA.hy926) and fibroblasts (NIH 3T3) intoxicated by Cd. The results revealed that EHN significantly improved the viability of EA.hy926 and NIH 3T3 cells, reduced apoptotic cell population, increased nitric oxide (NO) production and promoted blood vasculature development in the chick embryo model, which were hampered due to Cd insult. Molecular studies demonstrated the reduced expression of tumor suppressor (p53) and elevated anti-apoptotic protein (Bcl-xL) levels along with enhanced NO production through endothelial nitric oxide synthase (eNOS) activation as the plausible mechanisms underlying protective role of EHN against Cd-induced vascular toxicity. Considering the above observations, we strongly believe that EHN could be a potential nanomedicine approach for overcoming Cd-induced toxicity by improving vascular health and functioning.

Developing a partially perfused liver model using caprine liver explant conditioned to the chicken extra-embryonic perfusion system.

The Liver is constantly subjected to mechanical, chemical and pathological insults throughout life, as a result of which there is a common occurrence of various liver diseases. Due to the complex nature of liver architecture, it is not possible to mimic the in-vivo conditions beyond a certain limit. Hence, the development of in-vitro and ex-vivo models to study various liver diseases has gained more importance over the last few decades. The present study aims to develop a semi-perfused liver explant model to give an extended lifetime for studying liver pathology and treatment options. Caprine liver tissue explants were sliced, weighed (25 mg) and placed on the area vasculosa of fertilized chicken eggs. Unfertilized eggs were used as controls. After varying time intervals of incubation on area vasculosa of fertilized chicken eggs, the liver slices were subjected to cell viability assay, lactate dehydrogenase assay and Serum glutamic pyruvic transaminase assays. The results indicate that the viability and functional properties of such semi-perfused liver tissue explants holds good up to 6 h. Finally, the liver tissue explants were pre-treated with FBS, with and without anti-fibrotic drugs, and placed on chick embryo area vasculosa up to 6 h. The anti-fibrotic drug-treated semi perfused liver tissue explant showed a decrease in collagen formation as confirmed by histology and western blot. We deem that the use of extra-embryonic vasculature of chicken bed for extending the life of tissue explants will serve as a cost-effective alternative to animal models to understand disease mechanisms under drug treatments.

Differential expression of CRAC channel in alloxan induced Diabetic BALB/c mice.

Objectives: CRAC (Calcium Release Activated Calcium) channel is one of the most important channels regulating calcium influx and has been involved in many autoimmune diseases. The contribution of CRAC channel in the pathogenesis of Type 1 Diabetes (T1D) has not been described much. Thus, we aimed to study the expression of CRAC channel and inflammatory cytokines like IL-1ß (Interleukin -1ß) and TNF-α (Tumor Necrosis Factor-α) in the spleen-derived cytotoxic T cells, Bone marrow monocytes (BMM) and macrophages differentiated from BMM in the alloxan induced T1D mice.Materials and methods: BALB/c mice treated with alloxan and vehicle control for 12 and 24 h. Spleen derived T cells; Bone marrow derived monocytes were isolated from the control and diabetic BALB/c mice as well as macrophages differentiated from the control and diabetic BMM.Results: We observed increased expression of CRAC channel components like STIM1 (Stromal Interaction Molecule), ORAI1 and ORAI2 and inflammatory cytokines like IL-1ß and TNF-α in the spleen derived cytotoxic T cells and Macrophages differentiated from BMM as well as the downregulated expression of the same and CRAC channel in BMM of 12 and 24 h alloxan induced BALB/c mice.Conclusions: This study suggests that differential expression of CRAC channel correlated with the expression of inflammatory cytokines, thus CRAC channel might be responsible for the increased production of inflammatory cytokines in the alloxan induced T1D mice.

Nitric oxide restores peripheral blood mononuclear cell adhesion against hypoxia via NO-cGMP signalling.

Hypoxia is the most detrimental threat to humans residing at high altitudes, affecting multifaceted cellular responses that are crucial for normal homeostasis. Inhalation of nitric oxide has been successfully implemented to combat the hypoxia effect in the high altitude patients. We hypothesize that nitric oxide (NO) restores the peripheral blood mononuclear cell-matrix deadhesion during hypoxia. In the present study, we investigate the cellular action of exogenous NO in the hypoxia-mediated diminution of cell-matrix adhesion of PBMNC and NO bioavailability in vitro. The result showed that NO level and cell-matrix adhesion of PBMNC were significantly reduced in hypoxia as compared with normoxia, as assessed by the DAF-FM and cell adhesion assay, respectively. In contrast, cellular oxidative damage response was indeed upregulated in hypoxic PBMNC. Further, gene expression analysis revealed that mRNA transcripts of cell adhesion molecules (Integrin α5 and ß1) and eNOS expressions were significantly downregulated. The mechanistic study revealed that administration of NO and 8-Br-cGMP and overexpression of eNOS-GFP restored the basal NO level and recovers cell-matrix adhesion in PBMNC via cGMP-dependent protein kinase I (PKG I) signalling. In conclusion, NO-cGMP/PKG signalling may constitute a novel target to recover high altitude-afflicted cellular deadhesion. SIGNIFICANCE OF THIS STUDY: Cellular adhesion is a complex multistep process. The ability of cells to adhere to extracellular matrix is an essential physiological process for normal homeostasis and function. Hypoxia exposure in the PBMNC culture has been proposed to induce oxidative damage and cellular deadhesion and is generally believed to be the key factor in the reduction of NO bioavailability. In the present study, we demonstrated that NO donor or overexpression of eNOS-GFP has a protective effect against hypoxia-induced cellular deadhesion and greatly improves the redox balance by inhibiting the oxidative stress. Furthermore, this protective effect of NO is mediated by the NO-cGMP/PKG signal pathway, which may provide a potential strategy against hypoxia.