Desensitization of angiotensin receptor function

Desensitization of angiotensin receptor function. Angiotensin II is an eight amino acid peptide which plays a major role in the regulation of cardiovascular homeostasis. The physiologic effects of angiotensin (Ang) II are mediated by a G-protein coupled receptor, termed AT1, which activates phospholipase C. A major factor regulating angiotensin II receptor function is the rapid desensitization following agonist stimulation. However, despite years of investigation, the mechanism by which the angiotensin receptor is regulated remains unclear. The cloning of the AT-1 receptor and the availability of cell lines which stabily express this receptor has helped elucidate these mechanisms. In this paper, we review the data from our laboratory concerning the post-translational regulation of the angiotensin receptor function

901-90 In Vivo Genetic Engineering of Cardiac Cells: Intracoronary Administration of Antisense (AS) Oligonucleotides (ODN)

We have previously documented that transfection of antisense ODN by a highly efficient Sendai virus (HVJ)-liposome delivery system can be utilized to modify lesion formation within the peripheral vasculature in vivo. In this study, we defined the feasibility of modifying cardiac cell gene expression via a catheter-based coronary infusion of AS ODN in rabbits. The coronary artery was cannulated via an over-the-wire approach from the carotid artery. Fluorescein (F)-Iabeled ODN were utilized to evaluate the cellular distribution and kinetics of ODN uptake within the myocardium after a single intraluminal bolus of HVJ-liposomes containing ODN. Cellular uptake of F-ODN was primarily localized in the microvasculature and significant staining was also observed in conduit vessels and cardiac myocytes. Immunohistochemical analysis verified prominent localization of F-ODN within the microvascular endothelium. Expression of F-ODN was observed within 10 minutes, peaked at 1 day, and remained evident for up to one week after transfection by the HVJ-liposome method. In contrast, F-ODN infused within liposomes without the viral particle exhibited transient expression that was undetectable within 3 days. These findings indicate that a single intracoronary bolus infusion of ODN within HVJ-liposomes is a reproducible methodology for delivery of AS ODN to targeted cells within the myocardium. Future studies will characterize the feasibility of using this approach to modify cardiac structure and function via regulating myocardial cell gene expression

A cone-plate apparatus for the in vitro biochemical and molecular analysis of the effect of shear stress on adherent cells

Living cells are constantly exposed to a variety of complex mechanical stimuli which are though to be critical in the control of tissue structure and function. Endothelial and smooth muscle cells in the blood vessel are ideal candidates for the study of blood flow-induced cellular regulation. We describe here a cone-plate viscometer apparatus which is specially-designed for studying the effect of fluid shear stress on large populations of adherent cells in vitro. Using conventional polystyrene tissue culture plates, the apparatus is self-contained, fits inside a standard tissue culture incubator, and provides 75–150 cm 2 of useful surface area for cell growth. This capability makes it ideal for studying gene regulation using Northern analysis, nuclear runoff transcription, transfection with reporter constructs, as well as immunochemical staining. The closed-volume design of the device is also well-suited for isotopic labelling, pharmacological studies, and for the detection of minute amounts of secreted cell products. The setup allows the use of either steady, time- and direction-varying laminar, or turbulent shear stress. We provide a detailed assembly procedure and review the method for computing shear stress magnitude and Reynolds number. Ink flow analysis, dynamic response characterization, and LDH measurements are presented to confirm the device's fluid mechanical properties and demonstrate the absence of cell injury.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43234/1/11022_2004_Article_BF00996123.pd

Access to lifesaving medical resources for African countries: COVID-19 testing and response, ethics, and politics

Coronavirus disease 2019 (COVID-19) has revealed how strikingly unprepared the world is for a pandemic and how easily viruses spread in our interconnected world. A governance crisis is unfolding alongside the pandemic as health officials around the world compete for access to scarce medical supplies. As governments of African countries, and those in low-income and middle-income countries around the world, seek to avoid potentially catastrophic epidemics and learn from what has worked in other countries, testing and other medical resources are of concern. With accelerating spread, funding is urgently needed. Yet even where there is enough money, many African health authorities are unable to obtain the supplies needed as geopolitically powerful countries mobilise economic, political, and strategic power to procure stocks for their populations. We have seen this before. In the AIDS pandemic lifesaving diagnostics and drugs came to many African countries long after they were available in Europe and North America. In 2020, this situation can be avoided. Although health system weakness remains acute in many places, investments by national governments, the African Union, and international initiatives to tackle AIDS, tuberculosis, malaria, polio, and post-Ebola global health security have built important public health capacities. Global leaders have an ethical obligation to avoid needless loss of life due to the foreseeable prospect of slow and inadequate access to supplies in Africa

SFRP2 Regulates Cardiomyogenic Differentiation by Inhibiting a Positive Transcriptional Autofeedback Loop of Wnt3a

Wnts comprise a family of 20 lipid-modified glycoproteins in mammals and play critical roles during embryological development and organogenesis of several organ systems, including the heart. They are required for mesoderm formation and have been implicated in promoting cardiomyogenic differentiation of mammalian embryonic stem cells, but the underlying mechanisms regulating Wnt signaling during cardiomyogenesis remain poorly understood. In this report, we show that in a pluripotent mouse embryonal carcinoma stem cell line, SFRP2 inhibits cardiomyogenic differentiation by regulating Wnt3a transcription. SFRP2 inhibited early stages of cardiomyogenesis, preventing mesoderm specification and maintaining the cells in the undifferentiated state. Using a gain- and loss-of-function approach, we demonstrate that although addition of recombinant SFRP2 decreased Wnt3a transcription and cardiomyogenic differentiation, silencing of Sfrp2 led to enhanced Wnt3a transcription, mesoderm formation, and increased cardiomyogenesis. We show that the inhibitory effects of SFRP2 on Wnt transcription are secondary to interruption of a positive feedback effect of Wnt3a on its own transcription. Wnt3a increased its own transcription via the canonical pathway and TCF4 family of transcription factors, and the inhibitory effects of SFRP2 on Wnt3a transcription were associated with disruption of downstream canonical Wnt signaling. The inhibitory effects of Sfrp2 on Wnt3a expression identify Sfrp2 as a “checkpoint gene,” which exerts its control on cardiomyogenesis through regulation of Wnt3a transcription

MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes

Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ