Role of NADPH Oxidase-4 in human endothelial progenitor cells

Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization

Role of adrenergic receptors in shock

Shock is a severe, life-threatening medical condition with a high mortality rate worldwide. All four major categories of shock (along with their various subtypes)—hypovolemic, distributive, cardiogenic, and obstructive, involve a dramatic mismatch between oxygen supply and demand, and share standard features of decreased cardiac output, reduced blood pressure, and overall hypoperfusion. Immediate and appropriate intervention is required regardless of shock type, as a delay can result in cellular dysfunction, irreversible multiple organ failure, and death. Studies have shown that dysfunction and downregulation of adrenergic receptors (ARs) are often implicated in these shock conditions; for example, their density is shown to be decreased in hypovolemic and cardiogenic shock, while their reduced signaling in the brain and vasculature decrease blood perfusion and oxygen supply. There are two main categories of ARs, α, and β, each with its subtypes and distributions. Our group has demonstrated that a dose of .02 mg/kg body wt of centhaquine (CQ) specifically activates α2B ARs on venous circulation along with the central α2A ARs after hypovolemic/hemorrhagic shock. Activating these receptors by CQ increases cardiac output (CO) and reduces systemic vascular resistance (SVR), with a net increase in blood pressure and tissue perfusion. The clinical trials of CQ conducted by Pharmazz Inc. in India have demonstrated significantly improved survival in shock patients. CQ improved blood pressure and shock index, indicating better blood circulation, and reduced lactate levels in the blood compared to in-use standard resuscitative agents. After successful clinical trials, CQ is being marketed as a drug (Lyfaquin®) for hypovolemic/hemorrhagic shock in India, and United States FDA has approved the phase III IND application. It is anticipated that the phase III trial in the United States will begin in 2023. Thus, we have demonstrated that α2 ARs could be suitable targets for treating or managing hypovolemic/hemorrhagic shock. Further understanding of ARs in shock would help find new potential pharmacological targets

A Protocol for Measurement of Noncoding RNA in Human Serum

MicroRNAs (miRNAs) are small noncoding RNAs that act as regulators of gene expression by targeting mature messenger RNAs. Following the initial report of the presence of miRNAs in serum and plasma a number of studies have successfully demonstrated the use of these miRNAs as biomarkers of disease. Currently, there are many methods of isolating total RNA from liquid samples. Here, we describe a simple, cost effective method for extraction of RNA from human serum as well as subsequent real time PCR analysis of miRNA levels

Dynamics of Hot QCD Matter -- Current Status and Developments

The discovery and characterization of hot and dense QCD matter, known as Quark Gluon Plasma (QGP), remains the most international collaborative effort and synergy between theorists and experimentalists in modern nuclear physics to date. The experimentalists around the world not only collect an unprecedented amount of data in heavy-ion collisions, at Relativistic Heavy Ion Collider (RHIC), at Brookhaven National Laboratory (BNL) in New York, USA, and the Large Hadron Collider (LHC), at CERN in Geneva, Switzerland but also analyze these data to unravel the mystery of this new phase of matter that filled a few microseconds old universe, just after the Big Bang. In the meantime, advancements in theoretical works and computing capability extend our wisdom about the hot-dense QCD matter and its dynamics through mathematical equations. The exchange of ideas between experimentalists and theoreticians is crucial for the progress of our knowledge. The motivation of this first conference named "HOT QCD Matter 2022" is to bring the community together to have a discourse on this topic. In this article, there are 36 sections discussing various topics in the field of relativistic heavy-ion collisions and related phenomena that cover a snapshot of the current experimental observations and theoretical progress. This article begins with the theoretical overview of relativistic spin-hydrodynamics in the presence of the external magnetic field, followed by the Lattice QCD results on heavy quarks in QGP, and finally, it ends with an overview of experiment results.Comment: Compilation of the contributions (148 pages) as presented in the `Hot QCD Matter 2022 conference', held from May 12 to 14, 2022, jointly organized by IIT Goa & Goa University, Goa, Indi

Human Blood Vessel–Derived Endothelial Progenitors for Endothelialization of Small Diameter Vascular Prosthesis

BACKGROUND:Coronary bypass graft failure as a result of acute thrombosis and intimal hyperplasia has been the major challenge in surgical procedures involving small-diameter vascular prosthesis. Coating synthetic grafts with patients' own endothelial cells has been suggested to improve the patency rate and overall success of bypass surgeries. METHODOLOGY/PRINCIPAL FINDINGS:We isolated endothelial progenitor cells (EPCs) from leftover pieces of human saphenous vein/mammary artery. We demonstrate that EPCs can be expanded to generate millions of cells under low-density culture conditions. Exposure to high-density conditions induces differentiation to endothelial cell phenotype. EPC-derived endothelial cells show expression of CD144high, CD31, and vWF. We then assessed the ability of differentiated endothelial cells to adhere and grow on small diameter expanded polytetrafluoroethylene (ePTFE) tubings. Since ePTFE tubings are highly hydrophobic, we optimized protocols to introduce hydrophilic groups on luminal surface of ePTFE tubings. We demonstrate here a stepwise protocol that involves introduction of hydrophilic moieties and coating with defined ECM components that support adhesion of endothelial cells, but not of blood platelets. CONCLUSION/SIGNIFICANCE:Our data confirms that endothelial progenitors obtained from adult human blood vessels can be expanded in vitro under xenoprotein-free conditions, for potential use in endothelialization of small diameter ePTFE grafts. These endothelialized grafts may represent a promising treatment strategy for improving the clinical outcome of small-caliber vascular grafts in cardiac bypass surgeries

Sovateltide Mediated Endothelin B Receptors Agonism and Curbing Neurological Disorders

Neurological/neurovascular disorders constitute the leading cause of disability and the second leading cause of death globally. Major neurological/neurovascular disorders or diseases include cerebral stroke, Alzheimer’s disease, spinal cord injury, neonatal hypoxic-ischemic encephalopathy, and others. Their pathophysiology is considered highly complex and is the main obstacle in developing any drugs for these diseases. In this review, we have described the endothelin system, its involvement in neurovascular disorders, the importance of endothelin B receptors (ETBRs) as a novel potential drug target, and its agonism by IRL-1620 (INN—sovateltide), which we are developing as a drug candidate for treating the above-mentioned neurological disorders/diseases. In addition, we have highlighted the results of our preclinical and clinical studies related to these diseases. The phase I safety and tolerability study of sovateltide has shown it as a safe and tolerable compound at therapeutic dosages. Furthermore, preclinical and clinical phase II studies have demonstrated the efficacy of sovateltide in treating acute ischemic stroke. It is under development as a first-in-class drug. In addition, efficacy studies in Alzheimer’s disease (AD), acute spinal cord injury, and neonatal hypoxic-ischemic encephalopathy (HIE) are ongoing. Successful completion of these studies will validate that ETBRs signaling can be an important target in developing drugs to treat neurological/neurovascular diseases

Controls of Central and Peripheral Blood Pressure and Hemorrhagic/Hypovolemic Shock

The pressure exerted on the heart and blood vessels because of blood flow is considered an essential parameter for cardiovascular function. It determines sufficient blood perfusion, and transportation of nutrition, oxygen, and other essential factors to every organ. Pressure in the primary arteries near the heart and the brain is known as central blood pressure (CBP), while that in the peripheral arteries is known as peripheral blood pressure (PBP). Usually, CBP and PBP are correlated; however, various types of shocks and cardiovascular disorders interfere with their regulation and differently affect the blood flow in vital and accessory organs. Therefore, understanding blood pressure in normal and disease conditions is essential for managing shock-related cardiovascular implications and improving treatment outcomes. In this review, we have described the control systems (neural, hormonal, osmotic, and cellular) of blood pressure and their regulation in hemorrhagic/hypovolemic shock using centhaquine (Lyfaquin®) as a resuscitative agent

Sovateltide (IRL-1620) affects neuronal progenitors and prevents cerebral tissue damage after ischemic stroke

Stimulation of endothelin B receptors by its agonist IRL-1620 (INN, sovateltide) provides neuroprotection and neurological and motor function improvement following cerebral ischemia. We investigated the effect of sovateltide on stem and progenitor cells mediated neural regeneration and its effect on the cerebral tissue repair and restoration of neurological and motor function. Sovateltide (5 μg/kg) was injected intravenously in permanent middle cerebral artery occluded (MCAO) rats at 4, 6, and 8 h at days 0, 3, and 6. Neurological and motor function tests were carried out pre-MCAO and at day 7 post-MCAO. At day 7, significantly reduced expression of neuronal differentiation markers HuC/HuD and NeuroD1 was seen in MCAO + vehicle than sham rats. Sovateltide treatment upregulated HuC/HuD and NeuroD1 compared to MCAO + vehicle and their expression was similar to sham. Expression of stem cell markers Oct 4 and Sox 2 was similar in rats of all of the groups. Significantly reduced infarct volume and DNA damage with recovery of neurological and motor function was observed in sovateltide-treated MCAO rats. These results indicate that sovateltide initiates a regenerative response by promoting differentiation of neuronal progenitors and maintaining stem cells in an equilibrium following cerebral ischemic stroke.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Characterizing cellular identity at one cell resolution

Cellular processes are regulated at multiple levels in mammalian cells, including regulation at transcription, posttranscription, translation, and posttranslational levels. Most of the present techniques enable us to understand these processes either by analyzing RNAs or proteins. Very few methodologies such as combined in situ hybridization and immunocytochemistry allow us to visualize RNAs and proteins simultaneously in single cells. However, low abundance of certain transcripts (mRNAs and miRNAs) impedes the available methodologies to detect them at single-cell resolution. Here, we present a new improvised method of in situ TaqMan PCR in combination with immunostaining, which we developed to detect low abundance transcripts along with cellular proteins in mammalian cells. Cellular imaging carried out using confocal microscopy for proteins and RNAs (noncoding and messenger RNAs) can be analyzed simultaneously at single-cell resolution. The present method provides an enhanced understanding of the transcript and protein relationship at single-cell resolution and is especially useful to understand cellular populations that are highly heterogeneous

Exposure to morphine and caffeine induces apoptosis and mitochondrial dysfunction in a neonatal rat brain

Background: Preterm infants experience rapid brain growth during early post-natal life making them vulnerable to drugs acting on central nervous system. Morphine is administered to premature neonates for pain control and caffeine for apnea of prematurity. Simultaneous use of morphine and caffeine is common in the neonatal intensive care unit. Prior studies have shown acute neurotoxicity with this combination, however, little information is available on the mechanisms mediating the neurotoxic effects. The objective of this study was to determine the effects of morphine and caffeine, independently and in combination on mitochondrial dysfunction (Drp1 and Mfn2), neural apoptosis (Bcl-2, Bax, and cell damage) and endothelin (ET) receptors (ETA and ETB) in neonatal rat brain. Methods: Male and female rat pups were grouped separately and were divided into four different subgroups on the basis of treatments-saline (Control), morphine (MOR), caffeine (CAFF), and morphine + caffeine (M+C) treatment. Pups in MOR group were injected with 2 mg/kg morphine, CAFF group received 100 mg/kg caffeine, and M+C group received both morphine (2 mg/kg) and caffeine (100 mg/kg), subcutaneously on postnatal days (PND) 3-6. Pups were euthanized at PND 7, 14, or 28. Brains were isolated and analyzed for mitochondrial dysfunction, apoptosis markers, cell damage, and ET receptor expression via immunofluorescence and western blot analyses. Results: M+C showed a significantly higher expression of Bax compared to CAFF or MOR alone at PND 7, 14, 28 in female pups (p \u3c 0.05) and at PND 7, 14 in male pups (p \u3c 0.05). Significantly (p \u3c 0.05) increased expression of Drp1, Bax, and suppressed expression of Mfn2, Bcl-2 at PND 7, 14, 28 in all the treatment groups compared to the control was observed in both genders. No significant difference in the expression of ETA and ETB receptors in male or female pups was seen at PND 7, 14, and 28. Conclusion: Concurrent use of morphine and caffeine during the first week of life increases apoptosis and cell damage in the developing brain compared to individual use of caffeine and morphine