Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.

C-terminal variable AGES domain of Thymosin ß4: the molecule's primary contribution in support of post-ischemic cardiac function and repair.

Repairing defective cardiac cells is important towards improving heart function. Due to the frequency and severity of ischemic heart disease, management of patients featuring this type of cardiac failure receives significant interest. Previously we discovered that Thymosin ß4 (TB4), a 43 amino-acid secreted actin sequestering peptide, is beneficial for myocardial cell survival and coronary re-growth after infarction in adult mammals. Considering the regenerative potential of full-length TB4 in the heart, and that minimal structural variations alter TB4's influence on actin assembly and cell movement, we investigated how various TB4 domains affect cardiac cell behavior and post-ischemic mammalian heart function. We synthesized 17 domain combinations of full-length TB4 and analyzed their impact on embryonic cardiac cells in vitro, and after cardiac infarction in vivo. We discovered the domains of TB4 affect cardiac cell behavior distinctly. We revealed TB4 specific C-terminal tetrapeptide, AGES, increases embryonic cardiac cell migration and myocyte beating in culture, and improves adult mammalian heart function following ischemia. Investigating the molecular background and mechanism we discovered systemic injection of AGES enhances early myocyte survival by activating Akt-mediated signaling mechanisms, increases coronary vessel growth and inhibits inflammation in mice and pigs. Biodistribution analyses revealed cardiomyocytes uptake AGES efficiently in vitro and in vivo projecting a potential independent clinical utilization for the tetrapeptide. Our comprehensive domain investigations also suggest, preservation and/or restoration of cardiomyocyte communication is a target of TB4 and AGES, and critical to improve post-ischemic heart function in pigs. In summary, we identified the C-terminal four amino-acid variable end of TB4 as the essential and responsible domain for the molecule's full benefits in the hypoxic heart. Additionally, we introduced AGES as a novel, systemically applicable drug candidate to aid cardiac infarction in adult mammals.

Thymosin beta-4 - A potential tool in healing middle ear lesions in adult mammals.

Acute tympanic membrane perforations primarily occur due to injury or infection in humans. In acute cases, nearly 80-94 % of the perforations heal spontaneously. In chronic cases, non-surgical treatment becomes significantly limited, and the perforation can be restored only by myringoplasty. In addition to classical grafts such as the fascia or cartilage, promising results have been reported with various biological materials including silk or acellular collagen. However, despite of all the efforts, healing remains insufficient. Consequentially, a need for substances which actively promote tympanic cell migration and proliferation is deemed essential. In our study, we utilized Thymosin beta-4 (TB4), a 43aa peptide possessing many regenerative properties in various organ systems. Our aim was to reveal the impact of externally administered TB4 regarding impairments of the middle ear, particularly the tympanic membrane. We harvested tympanic membranes from adult mice and treated these with TB4 or PBS on both collagen gel matrixes and in the form of floating, ex vivo explants. Cell migration and proliferation was measured, while immunocytochemical analyses were performed to determine cell type and the nature of the targeted molecules. We discovered the peptide affects the behavior of epidermal and epithelial cells of the tympanic membrane in vitro. Moreover, as our initial results imply, it is not the differentiated, yet most likely the local epidermal progenitor cells which are the primary targets of the molecule. Our present results unveil a new, thus far undiscovered field regarding clinical utilization for TB4 in the future.

Thymosin beta-4 denotes new directions towards developing prosperous anti-aging regenerative therapies.

Our dream of defeating the processes of organ damage and aging remains a challenge scientists pursued for hundreds of years. Although the goal is to successfully treat the body as a whole, steps towards regenerating individual organs are even considered significant. Since initial approaches utilizing only progenitor cells appear limited, we propose interconnecting our collective knowledge regarding aging and embryonic development may lead to the discovery of molecules which provide alternatives to effectively reverse cellular damage. In this review, we introduce and summarize our results regarding Thymosin beta-4 (TB4) to support our hypothesis using the heart as model system. Accordingly, we investigated the developmental expression of TB4 in mouse embryos and determined the impact of the molecule in adult animals by systemically injecting the peptide following acute cardiac infarction or with no injury. Our results proved, TB4 is expressed in the developing heart and promotes cardiac cell migration and survival. In adults, the peptide enhances myocyte survival and improves cardiac function after coronary artery ligation. Moreover, intravenous injections of TB4 alter the morphology of the adult epicardium, and the changes resemble the characteristics of the embryo. Reactivation of the embryonic program became equally reflected by the increased number of cardiac vessels and by the alteration of the gene expression profile typical of the embryonic state. Moreover, we discovered TB4 is capable of epicardial progenitor activation, and revealed the effect is independent of hypoxic injury. By observing the above results, we believe, further discoveries and consequential postnatal administration of developmentally relevant candidate molecules such as TB4 may likely result in reversing aging processes and accelerate organ regeneration in the human body.

Utilizing Developmentally Essential Secreted Peptides Such as Thymosin Beta-4 to Remind the Adult Organs of Their Embryonic State-New Directions in Anti-Aging Regenerative Therapies.

Our dream of defeating the processes of aging has occupied the curious and has challenged scientists globally for hundreds of years. The history is long, and sadly, the solution is still elusive. Our endeavors to reverse the magnitude of damaging cellular and molecular alterations resulted in only a few, yet significant advancements. Furthermore, as our lifespan increases, physicians are facing more mind-bending questions in their routine practice than ever before. Although the ultimate goal is to successfully treat the body as a whole, steps towards regenerating individual organs are even considered significant. As our initial approach to enhance the endogenous restorative capacity by delivering exogenous progenitor cells appears limited, we propose, utilizing small molecules critical during embryonic development may prove to be a powerful tool to increase regeneration and to reverse the processes associated with aging. In this review, we introduce Thymosin beta-4, a 43aa secreted peptide fulfilling our hopes and capable of numerous regenerative achievements via systemic administration in the heart. Observing the broad capacity of this small, secreted peptide, we believe it is not the only molecule which nature conceals to our benefit. Hence, the discovery and postnatal administration of developmentally relevant agents along with other approaches may result in reversing the aging process.

Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair.

Heart disease is a leading cause of death in newborn children and in adults. Efforts to promote cardiac repair through the use of stem cells hold promise but typically involve isolation and introduction of progenitor cells. Here, we show that the G-actin sequestering peptide thymosin beta4 promotes myocardial and endothelial cell migration in the embryonic heart and retains this property in postnatal cardiomyocytes. Survival of embryonic and postnatal cardiomyocytes in culture was also enhanced by thymosin beta4. We found that thymosin beta4 formed a functional complex with PINCH and integrin-linked kinase (ILK), resulting in activation of the survival kinase Akt (also known as protein kinase B). After coronary artery ligation in mice, thymosin beta4 treatment resulted in upregulation of ILK and Akt activity in the heart, enhanced early myocyte survival and improved cardiac function. These findings suggest that thymosin beta4 promotes cardiomyocyte migration, survival and repair and the pathway it regulates may be a new therapeutic target in the setting of acute myocardial damage.

Thymosin beta4 mediated PKC activation is essential to initiate the embryonic coronary developmental program and epicardial progenitor cell activation in adult mice in vivo.

Hypoxic heart disease is a predominant cause of disability and death worldwide. Since adult mammalian hearts are incapable of regeneration after hypoxia, attempts to modify this deficiency are critical. As demonstrated in zebrafish, recall of the embryonic developmental program may be the key to success. Because thymosin beta4 (TB4) is beneficial for myocardial cell survival and essential for coronary development in embryos, we hypothesized that it reactivates the embryonic developmental program and initiates epicardial progenitor mobilization in adult mammals. We found that TB4 stimulates capillary-like tube formation of adult coronary endothelial cells and increases embryonic endothelial cell migration and proliferation in vitro. The increase of blood vessel/epicardial substance (Bves) expressing cells accompanied by elevated VEGF, Flk-1, TGF-beta, Fgfr-2, Fgfr-4, Fgf-17 and beta-Catenin expression and increase of Tbx-18 and Wt-1 positive myocardial progenitors suggested organ-wide recall of the embryonic program in the adult epicardium. TB4 also positively regulated the expression and phosphorylation of myristoylated alanine-rich C-kinase substrate (Marcks), a direct substrate and indicator of protein kinase C (PKC) activity in vitro and in vivo. PKC inhibition significantly reduced TB4 initiated epicardial thickening, capillary growth and the number of myocardial progenitors. Our results demonstrate that TB4 is the first known molecule capable of organ-wide activation of the embryonic coronary developmental program in the adult mammalian heart after systemic administration and that PKC plays a significant role in the process.

Thymosin beta4 is an essential paracrine factor of embryonic endothelial progenitor cell-mediated cardioprotection.

BACKGROUND: Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin beta4 (Tbeta4) as a mediator of eEPC-mediated cardioprotection. METHODS AND RESULTS: In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tbeta4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5x10(6) cells) or cells transfected with Tbeta4 shRNA when indicated or 15 mg Tbeta4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32+/-4% to 90+/-2% after eEPC application, an effect sensitive to shRNA transfection compared with Tbeta4 (45+/-7%). In vivo, infarct size decreased with eEPC application (38+/-4% versus 54+/-4% of area at risk; P<0.01), an effect abolished by Tbeta4 shRNA (62+/-3%). Segmental subendocardial shortening improved after eEPC treatment (22+/-3% versus -3+/-4% of control area) unless Tbeta4 shRNA was transfected (-6+/-4%). Retroinfusion of Tbeta4 mimicked eEPC application (infarct size, 37+/-3%; segmental endocardial shortening, 34+/-7%). Myeloperoxidase activity (3323+/-388 U/mg in controls) was decreased by eEPCs (1996+/-546 U/mg) or Tbeta4 alone (1455+/-197 U/mg) but not Tbeta4 shRNA-treated eEPCs (5449+/-829 U/mg). CONCLUSIONS: Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to Tbeta4.

Serologic survey of the Crimean-Congo haemorrhagic fever virus infection among wild rodents in Hungary.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne pathogen, which causes an increasing number of severe infections in many parts of Africa, Asia and in Europe. The virus is primarily transmitted by ticks, however, the spectrum of natural hosts regarding CCHFV includes a wide variety of domestic and wild animals. Although the presence of CCHFV was hypothesized in Hungary, data in support of CCHFV prevalence has thus far, proven insufficient. In the present study, rodents belonging to four species, the yellow-necked mouse (Apodemus flavicollis), the striped field mouse (A. agrarius), the wood mouse (A. sylvaticus) and the bank vole (Myodes glareolus), were all systematically trapped in the Mecsek Mountain region (Southwest Hungary), from 2011 through 2013. Rodent sera were collected and screened for CCHFV antibodies with dot-blot pre-screening and immunofluorescence assay. Among the 2085 tested rodents, 20 (0.96%) were positive for IgG antibody against CCHFV. Seroprevalence was the highest (1.25%) in A. flavicollis serum samples. Distinctly, we now provide the first data regarding CCHFV occurrence and seroprevalence among wild rodents in Hungary. This observation represents a need for large-scale surveillance to effectively assess the enzootic background and the potential public health risk of CCHFV in Hungary.

Thymosin beta4 is cardioprotective after myocardial infarction.

Heart disease is a leading cause of death in newborns and in adults. Efforts to promote cardiac repair by introduction or recruitment of exogenous stem cells hold promise but typically involve isolation and introduction of autologous or donor progenitor cells. We have found that the G-actin-sequestering peptide thymosin beta4 promotes myocardial and endothelial cell migration in the embryonic heart and retains this property in postnatal cardiomyocytes. Survival of embryonic and postnatal cardiomyocytes in culture was also enhanced by thymosin beta4. We found that thymosin beta4 formed a functional complex with PINCH and integrin-linked kinase (ILK), resulting in activation of the survival kinase Akt/PKB, which was necessary for thymosin beta4's effects on cardiomyocytes. After coronary artery ligation in mice, thymosin beta4 treatment resulted in upregulation of ILK and Akt activity in the heart, enhanced early myocyte survival, and improved cardiac function. These findings suggest that thymosin beta4 promotes cardiomyocyte and endothelial migration, survival, and repair and may be a novel therapeutic target in the setting of acute myocardial damage.

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2.

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin ß4 (Tß4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or Tß4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the Tß4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as Tß4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing Tß4 (Tß4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

Thymosin beta4 and cardiac repair.

Hypoxic heart disease is a predominant cause of disability and death worldwide. As adult mammals are incapable of cardiac repair after infarction, the discovery of effective methods to achieve myocardial and vascular regeneration is crucial. Efforts to use stem cells to repopulate damaged tissue are currently limited by technical considerations and restricted cell potential. We discovered that the small, secreted peptide thymosin beta4 (Tbeta4) could be sufficiently used to inhibit myocardial cell death, stimulate vessel growth, and activate endogenous cardiac progenitors by reminding the adult heart on its embryonic program in vivo. The initiation of epicardial thickening accompanied by increase of myocardial and epicardial progenitors with or without infarction indicate that the reactivation process is independent of injury. Our results demonstrate Tbeta4 to be the first known molecule able to initiate simultaneous myocardial and vascular regeneration after systemic administration in vivo. Given our findings, the utility of Tbeta4 to heal cardiac injury may hold promise and warrant further investigation.

Thymosin beta4: a key factor for protective effects of eEPCs in acute and chronic ischemia.

Acute myocardial infarction is still one of the leading causes of death in the industrial nations. Even after successful revascularization, myocardial ischemia results in a loss of cardiomyocytes and scar formation. Embryonic EPCs (eEPCs), retroinfused into the ischemic region of the pig heart, provided rapid paracrine benefit to acute and chronic ischemia in a PI-3K/Akt-dependent manner. In a model of acute myocardial ischemia, infarct size and loss of regional myocardial function decreased after eEPC application, unless cell pre-treatment with thymosin beta4 shRNA was performed. Thymosin beta4 peptide retroinfusion mimicked the eEPC-derived improvement of infarct size and myocardial function. In chronic ischemia (rabbit model), eEPCs retroinfused into the ischemic hindlimb enhanced capillary density, collateral growth, and perfusion. Therapeutic neovascularization was absent when thymosin beta4 shRNA was introduced into eEPCs before application. In conclusion, eEPCs are capable of acute and chronic ischemia protection in a thymosin beta4 dependent manner.

Embryonic endothelial progenitor cell-mediated cardioprotection requires Thymosin beta4.

Myocardial damage is frequently occurring upon a prolonged period of ischemia, although subsequent reperfusion as standard therapy is established. Among the pleiotropic causes of ischema-reperfusion injury, loss of cardiomyocytes, microcirculatory disturbances, and postischemic inflammation have been frequently observed. Current clinical cell therapy after acute myocardial mostly aims at neovascularization and enhancement of tissue repair, whereas acute cardioprotection after ischemia and reperfusion has rarely been studied. Recently, embryonic endothelial progenitor cells (eEPCs) have been found to provide cardioprotection against acute ischemia-reperfusion injury (24 hours) in a preclinical pig model. The paracrine effect of eEPCs was mimicked by regional application of a single, highly expressed protein, Thymosin beta4. This review focuses on underlying mechanisms of acute cardioprotection provided by eEPCs and, in particular, Thymosin beta4.