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MG53, a member of the tripartite motif (TRIM) protein family, plays an essential role in cell membrane repair and promotes cell survival. Recent studies show that systemic delivery of recombinant human MG53 (rhMG53) protein markedly attenuates tissue injury/inflammation, and facilitates healing. This study was performed to test whether intravenous administration of rhMG53 protein would decrease the lesion size in a clinically relevant large animal model of traumatic brain injury (TBI).Yorkshire swine (40-45 kg; n = 5/group) were subjected to controlled cortical impact TBI and randomized to either: (1) rhMG53 protein (2 mg/kg, intravenous) or (2) normal saline control. Hemodynamics, intracranial pressure, and brain oxygenation were monitored for 7 hours. Brains were then harvested and sectioned into 5-mm slices and stained with 2,3,5-triphenyltetrazolium chloride to quantify the lesion size. Blood-brain barrier permeability of MG53 in the brain was determined by Western blot and immunohistochemistry. Bcl-2 and phospho-GSK β levels were measured as makers of prosurvival pathway activation.Hemodynamic parameters were similar in both groups, but the lesion size in the rhMG53-treated group (2,517 ± 525.4 mm 3 ) was significantly ( p0.05) smaller than the control group (3,646 ± 740.1 mm 3 ). In the treated animals, rhMG53 was detected in the regions surrounding the TBI, but it was absent in the saline-treated control animals. Bcl-2 and phospho-GSK β levels in the brains were upregulated in the rhMG53-treated animals.Intravenously administered rhMG53 localizes to the injured areas of the brain, with the treated animals demonstrating a significant attenuation in the brain lesion size following TBI.

Nitrogen mustard (NM) is an alkylating vesicant that causes severe pulmonary injury. Currently, there are no effective means to counteract vesicant-induced lung injury. MG53 is a vital component of cell membrane repair and lung protection. Here, we show that mice with ablation of MG53 are more susceptible to NM-induced lung injury than the wild-type mice. Treatment of wild-type mice with exogenous recombinant human MG53 (rhMG53) protein ameliorates NM-induced lung injury by restoring arterial blood oxygen level, by improving dynamic lung compliance and by reducing airway resistance. Exposure of lung epithelial and endothelial cells to NM leads to intracellular oxidative stress that compromises the intrinsic cell membrane repair function of MG53. Exogenous rhMG53 protein applied to the culture medium protects lung epithelial and endothelial cells from NM-induced membrane injury and oxidative stress, and enhances survival of the cells. Additionally, we show that loss of MG53 leads to increased vulnerability of macrophages to vesicant-induced cell death. Overall, these findings support the therapeutic potential of rhMG53 to counteract vesicant-induced lung injury.

Influenza virus activates cellular inflammasome pathways, which can be either beneficial or detrimental to infection outcomes. Here, we investigated the role of the inflammasome-activated pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice significantly attenuated virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected GSDMD KO mice exhibited decreased inflammatory gene signatures revealed by lung transcriptomics, which also implicated a diminished neutrophil response. Importantly, neutrophil depletion in infected WT mice recapitulated the reduced mortality and lung inflammation observed in GSDMD KO animals, while having no additional protective effects in GSDMD KOs. These findings reveal a new function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a new therapeutic avenue for treating severe influenza.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with limited therapeutic options and a dismal long-term survival. The upregulation of KIF11, a motor protein that regulates cell mitosis, has been associated with the aggressive proliferation of pancreatic cancer cells. A molecular strategy to counteract the hyperactivity of KIF11 may represent a potential means to treat PDAC. We previously identified MG53, a member of the tripartite motif family protein (TRIM72), as an important component of tissue repair, and revealed its potential application in regenerative medicine. So far, most published studies have focused on the cytosolic and vesicle-trafficking function of MG53, little is known on the nuclear action of MG53 in pancreatic tumor suppression. In the present study, our confocal imaging revealed frequent appearance of MG53 in the nucleus of human pancreatic cancer cells, and subcellular fractionation assay showed that a majority of MG53 protein was present in the nuclear fraction. Decreased MG53 and increased KIF11 protein expression were observed in both pancreatic cancer cell lines (PANC-1 and mPanc96) and PDAC tissue from surgically resected tumors. Overexpressing MG53 inhibited colony formation and proliferation of PANC-1 cells with significant reduction of KIF11 expression. Furthermore, we demonstrated that MG53-mediated control of KIF11 expression occurs at the gene transcription level. Chromatin immunoprecipitation demonstrated an interaction between MG53 and the promoter of KIF11 in PANC-1 cells. Using a luciferase cell reporter driven by the KIF11 promoter, we found that overexpressing MG53 led to downregulation of the KIF11 transcription activity, and induced cell cycle arrest at the G2/M phase. We also conducted syngeneic orthotopic transplantation of a mouse pancreatic cancer cell line, KPC cells derived from pancreatic tumor in KRasG12D/Trp53-/-/Pdx-1-Cre (KPC) mice, into the pancreas of wild type (WT) and mg53-/- (MG53-KO) mice, which showed aggressive pancreatic tumor growth in MG53-KO mice compared with WT mice after 3-week of transplantation. Western blots revealed that a considerable amount of MG53 protein was found in the pancreatic tumors derived from KPCs in WT mice vs. those in MG53-KO mice, indicating the accumulation of circulating MG53 during tumor growth. Overall, these findings demonstrated that MG53 functions as a tumor suppressor to inhibit cancer cell proliferation and the tumor growth through transcriptional down-regulating the expression of KIF11 in pancreatic cancer, and provided a new therapeutic target for the treatment of patients with PDAC. Citation Format: Xiao-Liang Wang, Xiangfei He, Zobeida Cruz-Monserrate, Allen Tsung, Jianjie Ma, Chuanxi Cai. MG53 suppresses the tumor growth via transcriptional inhibiting the expression of KIF11 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1729.

Introduction Lung injury has several inciting etiologies ranging from trauma (contusion and hemorrhage) to ischemia reperfusion injury. Reflective of the injury, tissue and cellular injury increases proportionally with the injury stress and is an area of potential intervention to mitigate the injury. This study aims to evaluate the therapeutic benefits of recombinant human MG53 (rhMG53) protein in porcine models of acute lung injury (ALI). Materials and Methods We utilized live cell imaging to monitor the movement of MG53 in cultured human bronchial epithelial cells following mechanical injury. The in vivo efficacy of rhMG53 was evaluated in a porcine model of hemorrhagic shock/contusive lung injury. Varying doses of rhMG53 (0, 0.2, or 1 mg/kg) were administered intravenously to pigs after induction of hemorrhagic shock/contusive induced ALI. Ex vivo lung perfusion system enabled assessment of the isolated porcine lung after a warm ischemic induced injury with rhMG53 supplementation in the perfusate (1 mg/mL). Results MG53-mediated cell membrane repair is preserved in human bronchial epithelial cells. rhMG53 mitigates lung injury in the porcine model of combined hemorrhagic shock/contusive lung injury. Ex vivo lung perfusion administration of rhMG53 reduces warm ischemia-induced injury to the isolated porcine lung. Conclusions MG53 is an endogenous protein that circulates in the bloodstream. Therapeutic treatment with exogenous rhMG53 may be part of a strategy to restore (partially or completely) structural morphology and/or functional lung integrity. Systemic administration of rhMG53 constitutes a potential effective therapeutic means to combat ALI.

MG53 is a member of the TRIM protein family that is predominantly expressed in striated muscles and participates in cell membrane repair. Controversy exists regarding MG53’s role in insulin signaling and manifestation of diabetes. We generated db/db mice with either whole-body ablation or sustained elevation of MG53 in the bloodstream in order to evaluate the physiological function of MG53 in diabetes. To quantify the amount of MG53 protein in circulation, we developed a monoclonal antibody against MG53 with high specificity. Western blot using this antibody revealed lower or no change of serum MG53 levels in db/db mice or patients with diabetes compared with control subjects. Neither whole-body ablation of MG53 nor sustained elevation of MG53 in circulation altered insulin signaling and glucose handling in db/db mice. Instead, mice with ablation of MG53 were more susceptible to streptozotocin-induced dysfunctional handling of glucose compared with the wild-type littermates. Alkaline-induced corneal injury demonstrated delayed healing in db/db mice, which was restored by topical administration of recombinant human (rh)MG53. Daily intravenous administration of rhMG53 in rats at concentrations up to 10 mg/kg did not produce adverse effects on glucose handling. These findings challenge the hypothetical function of MG53 as a causative factor for the development of diabetes. Our data suggest that rhMG53 is a potentially safe and effective biologic to treat diabetic oculopathy in rodents.

Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.

Introduction: Aging is associated with chronic oxidative stress and inflammation that affect the tissue repair and regeneration capacity. MG53 is a TRIM family protein that facilitates repair of cell membrane injury in a redox-dependent manner. However, it is not clear how MG53 regulates cardiac inflammation during aging. Hypothesis: We hypothesize that the expression of MG53 is associated with age-related heart failure, and treatment with recombinant human MG53 (rhMG53) protein in aged mice might have beneficial effect on the age-related decline of heart function. Methods: qPCR and Western blots were conducted for targeted genes/proteins in non-failing and failing human left ventricular tissues, and mouse heart tissues. A total of sixty mice (24 months, 30 male and 30 female) were enrolled for treatment with rhMG53 (6 mg/kg, subcutaneous, daily for 6 weeks) or saline as control, and subjected to cardiac functional measurement with echocardiography and pressure-volume loop, and cardiac apoptotic and ROS assays. Blood were collected to quantify the serum levels of biomarkers for liver and cardiovascular injury. Results: We demonstrate that the expression of MG53 is reduced in failing human heart and aging mouse heart, concomitant with elevated NFκB activation. We evaluate the safety and efficacy of longitudinal, systemic administration of recombinant human MG53 (rhMG53) protein in aged mice. Echocardiography and pressure-volume loop measurements reveal beneficial effects of rhMG53 treatment in improving heart function of aging mice. Biochemical and histological studies demonstrate the cardioprotective effects of rhMG53 are linked to suppression of NFκB-mediated inflammation, reducing apoptotic cell death and oxidative stress in the aged heart. Repetitive administration of rhMG53 in aged mice does not have adverse effects on immune response and major vital organ functions. Conclusions: In this study, we showed decreased expression of MG53 and increased NFκB activation in both failing human hearts and aging mouse hearts. Moreover, longitudinal administration of rhMG53 mitigated the heart dysfunction in the aged mice. These findings support the therapeutic value of rhMG53 in treating age-related decline in cardiac function.

Cardiac dysfunction is a common extrapulmonary complication of severe influenza virus infection. Prevailing models propose that influenza-associated heart dysfunction is indirectly triggered by cytokine mediated cardiotoxicity downstream of the inflamed lung, rather than by direct infection of cardiac tissue. To test the etiology of cardiac dysfunction resulting from influenza virus infection, we generated a novel recombinant H1N1 influenza A virus that was attenuated in cardiomyocytes by incorporation of target sequences for miRNAs expressed specifically in that cell type (miR133b and miR206). Compared with control virus, mice infected with the miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. The miR-targeted virus, however, was fully replicative and inflammatory in lungs when compared to control virus, and induced similar systemic weight loss. The miR-targeted virus induced considerably lower levels of cardiac arrhythmia, fibrosis, and inflammation, compared with control virus, in mice lacking interferon induced transmembrane protein 3 (IFITM3), which serve as the only available model for severe influenza-associated cardiac pathology. We conclude that robust replication of virus in the heart is required for pathology even when lung inflammation is severe. Indeed, we show that human stem cell-derived cardiomyocytes are susceptible to influenza virus infection. This work establishes a fundamental new paradigm in which influenza virus damages the heart through direct infection of cardiomyocytes.

Cancer is the second leading cause of mortality after cardiovascular diseases in the United States. Chemotherapy is widely used to treat cancers. Since the development of drug resistance is a major contributor towards the failure of chemotherapeutic regimens, efforts have been made to develop novel inhibitors that can combat drug resistance and sensitize cancer cells to chemotherapy. Here we investigated the anti-cancer effects of MG53, a TRIM-family protein known for its membrane repair functions. We found that rhMG53 reduced cellular proliferation of both parental and ABCB1 overexpressing colorectal carcinoma cells. Exogenous rhMG53 protein entered SW620 and SW620/AD300 cells without altering the expression of ABCB1 protein. In a mouse SW620/AD300 xenograft model, the combination of rhMG53 and doxorubicin treatment significantly inhibited tumor growth without any apparent weight loss or hematological toxicity in the animals. Our data show that MG53 has anti-proliferative function on colorectal carcinoma, regardless of their nature to drug-resistance. This is important as it supports the broader value for rhMG53 as a potential adjuvant therapeutic to treat cancers even when drug-resistance develops.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

Background: Mitochondrial dysfunction is an important risk factor for heart failure in elderly people. Mitophagy, a physiological process that controls the removal of damaged mitochondria, is compromised in aging or failing hearts. In this study, we examined the physiological role of a cardiac-specific lncRNA Camirt that can potentially modulate mitophagy in the heart. Methods and Results: RNA-seq analysis and RT-PCR reveal a lncRNA is highly expressed in both mouse and human hearts, with undetectable levels in other vital organs. Furthermore, Real time qPCR was used to examine the expression of lncRNA in different animal models and in human hearts, which results showed that the expression of this lncRNA is decreased in aging mouse and human hearts, and failing mouse hearts induced by isoproterenol and doxorubicin. RNA pull-down and RNA-binding protein immunoprecipitation assays identify prohibitin-2 (Phb2), a known mitophagy receptor, as a binding partner for this lncRNA. Thus, we name this novel lncRNA as a cardiac-specific mitophagy-associated RNA transcript (Camirt). Camirt conditional (flox) knockout mice were created via CRISPR /Cas9 technology, and subjected to the longitudinal echocardiographic and survival studies. Mice with cardiac specific deletion of Camirt (Camirt-cKO) display progressive heart failure and die within 12 month after birth. RNA sequencing and gene ontology analysis revealed that genes involved in mitophagy signaling were significantly altered in the Camirt-cKO hearts compared with the littermate wild type mice. Transmission electron microscopy were used to examine the mitochondrial morphology in mouse hearts, and reveal excessive accumulation of mitolysosomes in cardiomyocytes derived from the Camirt-cKO mice. In vitro study with Annexin-V/PI staining showed an increased number of live cells and decreased number of apoptotic cells in cultured neonatal cardiomyocytes with overexpression of Camirt following oxidative stress induced by treatment with H2O2. Increased autophagy (or mitophagy) activity was observed in HL-1 cells with stable overexpression of Camirt and in the presence of chloroquine (an inhibitor for the lysosome degradation). While reduced Camirt expression via shRNA knock-down leads to compromised mitophagy activity in HL-1 cells. Further biochemical studies support the function of Camirt/Phb2 in maintenance of mitochondria function and mitophagy signaling under stress conditions. Conclusion: Overall, our results suggested that Camirt plays a sentinel role in aging-related heart failure via interaction with Phb2 to modulate mitophagy signaling in the heart. Future studies will focus on elucidating the in vivo role and mechanisms of Camirt in modulation of mitophagy under natural aging or stress-induced pathologic conditions using the loss- or gain-of-function of Camirt mouse models.

Heart disease remains the leading cause of mortality globally, so further investigation is required to identify its underlying mechanisms and potential targets for treatment and prevention. Mitsugumin 53 (MG53), also known as TRIM72, is a TRIM family protein that was found to be involved in cell membrane repair and primarily found in striated muscle. Its role in skeletal muscle regeneration and myogenesis has been well documented. However, accumulating evidence suggests that MG53 has a potentially protective role in heart tissue, including in ischemia/reperfusion injury of the heart, cardiomyocyte membrane injury repair, and atrial fibrosis. This review summarizes the regulatory role of MG53 in cardiac tissues, current debates regarding MG53 in diabetes and diabetic cardiomyopathy, as well as highlights potential clinical applications of MG53 in treating cardiac pathologies.

Significance Measles virus (MeV) vaccine is one of the safest and most efficient vaccines with a track record in children. Here, we generated a panel of rMeV-based vaccines with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S antigens inserted near 3′ of the MeV genome. The rMeV expressing a soluble stabilized, prefusion spike (preS) is much more potent in triggering SARS-CoV-2–specific neutralizing antibody than rMeV-based full-length S vaccine candidate. A single dose of rMeV-preS is sufficient to induce high levels of SARS-CoV-2 antibody in animals. Furthermore, rMeV-preS induces high levels of Th1-biased immunity. Hamsters immunized with rMeV-preS were completely protected against SARS-CoV-2 challenge. Our results demonstrate rMeV-preS is a safe and highly efficacious bivalent vaccine candidate for SARS-CoV-2 and MeV., The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR−/−mice, IFNAR−/−-hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2–specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

Aging is associated with chronic oxidative stress and inflammation that affect tissue repair and regeneration capacity. MG53 is a TRIM family protein that facilitates repair of cell membrane injury in a redox-dependent manner. Here, we demonstrate that the expression of MG53 was reduced in failing human hearts and aged mouse hearts, concomitant with elevated NF-κB activation. We evaluated the safety and efficacy of longitudinal, systemic administration of recombinant human MG53 (rhMG53) protein in aged mice. Echocardiography and pressure-volume loop measurements revealed beneficial effects of rhMG53 treatment in improving heart function of aged mice. Biochemical and histological studies demonstrated that the cardioprotective effects of rhMG53 are linked to suppression of NF-κB-mediated inflammation, reducing apoptotic cell death and oxidative stress in the aged heart. Repetitive administration of rhMG53 in aged mice did not have adverse effects on major vital organ functions. These findings support the therapeutic value of rhMG53 in treating age-related decline in cardiac function.

An intrusion prevention system (IPS) acts as a new type of information security technology, the configuration and management of which are currently urgent problems; in particular, debate exists regarding the value of these systems. In this paper, we analyse whether a firm realizes a positive or negative value from using an IPS instead of an intrusion detection system (IDS) in a default configuration and an optimal configuration, respectively. Our results suggest: (a) an IPS could hurt the firm when not configured optimally; (b) the optimal configuration of the IPS depends not only on the cost parameters but also on the external environment (quality of the IDS) in which the firm is operating; (c) whether the IDS is optimally configured or not, the firm will make the same decisions between using the IPS instead of the IDS and continuing to use the IDS; and (d) except for the true positive rate of IDS being in a certain region and the blocking cost being sufficiently high, the firm realizes a strictly nonnegative value if the firm configures the IPS optimally.

Cardiac stem/progenitor cells (CPCs) have recently emerged as a potentially transformative regenerative medicine to repair the infarcted heart. However, the limited survival of donor cells is one of the major challenges for CPC therapy. Our recent research effort on preconditioning human CPCs (hCPCs) with cobalt protoporphyrin (CoPP) indicated that sulfiredoxin-1 (SRXN1) is upregulated upon preconditioning aldehyde dehydrogenase bright hCPCs (ALDH(br)-hCPCs) with CoPP. Further studies demonstrated that overexpressing SRXN1 enhanced the survival capacity for ALDH(br)-hCPCs. This was associated with the up-regulation of anti-apoptotic factors, including BCL2 and BCL-xL. Meanwhile, overexpressing SRXN1 decreased the ROS generation and mitochondrial membrane potential, concomitant with the up-regulated primary antioxidant systems, such as PRDX1, PRDX3, TXNRD1, Catalase and SOD2. It was also observed that overexpressing SRXN1 increased the migration, proliferation, and cardiac differentiation of ALDH(br)-hCPCs. Interestingly, SRXN1 activated the ERK/NRF2 cell survival signaling pathway, which may be the underlying mechanism through which overexpressing SRXN1 lead to protection of hCPCs against oxidative stress-induced apoptosis. Taken together, these results provide a rationale for the exploration of SRXN1 as a novel molecular target that can be used to enhance the effectiveness of cardiac stem/progenitor cell therapy for ischemic heart disease.

Human cardiac stem/progenitor cells (hCPCs) may serve in regenerative medicine to repair the infarcted heart. However, this approach is severely limited by the poor survival of donor cells. Recent studies suggest that the mammalian globin cytoglobin (CYGB) regulates nitric oxide (NO) metabolism and cell death. In the present study, we found that CYGB is expressed in hCPCs. Through molecular approaches aimed at increasing or decreasing CYGB expression in hCPCs, we found that CYGB functions as a pro-survival factor in response to oxidative stress. This was associated with the upregulation of primary antioxidant systems such as peroxiredoxins-1, heme oxygenase-1, and anti-apoptotic factors, including BCL2, BCL-XL, and MCL1. Most significantly, we established that CYGB increased the expression of NFкB-dependent genes including iNOS, and that iNOS-dependent NO production was required for a feedforward loop that maintains CYGB expression. Our study delineates for the first time a role for a globin in regulating hCPC survival and establishes mechanistic insights in the function of CYGB. It provides a rationale for the exploration of the CYGB pathway as a molecular target that can be used to enhance the effectiveness of cardiac stem/progenitor cell therapy for ischemic heart disease.

MG53 is a member of the TRIM protein family that is predominantly expressed in striated muscles and participates in cell membrane repair. Controversy exists regarding MG53’s role in insulin signaling and manifestation of diabetes. We generated db/db mice with either whole-body ablation or sustained elevation of MG53 in the bloodstream in order to evaluate the physiological function of MG53 in diabetes. To quantify the amount of MG53 protein in circulation, we developed a monoclonal antibody against MG53 with high specificity. Western blot using this antibody revealed lower or no change of serum MG53 levels in db/db mice or patients with diabetes compared with control subjects. Neither whole-body ablation of MG53 nor sustained elevation of MG53 in circulation altered insulin signaling and glucose handling in db/db mice. Instead, mice with ablation of MG53 were more susceptible to streptozotocin-induced dysfunctional handling of glucose compared with the wild-type littermates. Alkaline-induced corneal injury demonstrated delayed healing in db/db mice, which was restored by topical administration of recombinant human (rh)MG53. Daily intravenous administration of rhMG53 in rats at concentrations up to 10 mg/kg did not produce adverse effects on glucose handling. These findings challenge the hypothetical function of MG53 as a causative factor for the development of diabetes. Our data suggest that rhMG53 is a potentially safe and effective biologic to treat diabetic oculopathy in rodents.

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H(2)O(2), concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

Myocardial infarctions and chronic ischemic heart disease both commonly and disproportionately affect elderly patients more than any other patient population. Despite available treatments, heart tissue is often permanently damaged as a result of cardiac injury. This review aims to summarize recent literature proposing the use of modified autologous adult stem cells to promote healing of post-infarct cardiac tissue. This novel cellular treatment involves isolation of adult stem cells from the patient, in vitro manipulation of these stem cells, and subsequent transplantation back into the patient's own heart to accelerate healing. One of the hindrances affecting this process is that cardiac issues are increasingly common in elderly patients, and stem cells recovered from their tissues tend to be pre-senescent or already in senescence. As a result, harsh in vitro manipulations can cause the aged stem cells to undergo massive in vivo apoptosis after transplantation. The consensus in literature is that inhibition or reversal of senescence onset in adult stem cells would be of utmost benefit. In fact, it is believed that this strategy may lower stem cell mortality and coerce aged stem cells into adopting more resilient phenotypes similar to that of their younger counterparts. This review will discuss a selection of the most efficient and most-recent strategies used experimentally to enhance the effectiveness of current stem cell therapies for ischemic heart diseases.

Cardiac stem cell therapy has shown very promising potential to repair the infarcted heart but is severely limited by the poor survival of donor cells. Nitric oxide (NO) has demonstrated cytoprotective properties in various cells, but its benefits are unknown specifically for human cardiac stem cells (hCSCs). Therefore, we investigated whether pretreatment of hCSCs with a widely used NO donor, diethylenetriamine nitric oxide adduct (DETA-NO), promotes cell survival. Results from lactate dehydrogenase release assays showed a dose- and time-dependent attenuation of cell death induced by oxidative stress after DETA-NO preconditioning; this cytoprotective effect was abolished by the NO scavenger. Concomitant up-regulation of several cell signaling molecules after DETA-NO preconditioning was observed by Western blotting, including elevated phosphorylation of NRF2, NFκB, STAT3, ERK, and AKT, as well as increased protein expression of HO-1 and COX2. Furthermore, pharmaceutical inhibition of ERK, STAT3, and NFκB activities significantly diminished NO-induced cytoprotection against oxidative stress, whereas inhibition of AKT or knockdown of NRF2 only produced a minor effect. Blocking PI3K activity or knocking down COX2 expression did not alter the protective effect of DETA-NO on cell survival. The crucial roles of STAT3 and NFκB in NO-mediated signaling pathways were further confirmed by stable expression of gene-specific shRNAs in hCSCs. Thus, preconditioning hCSCs with DETA-NO promotes cell survival and resistance to oxidative stress by activating multiple cell survival signaling pathways. These results will potentially provide a simple and effective strategy to enhance survival of hCSCs after transplantation and increase their efficacy in repairing infarcted myocardium.

Autologous human cardiac stem/progenitor cell (hCPC) therapy is a promising treatment that has come into use in recent years for patients with cardiomyopathy. Though innovative in theory, a major hindrance to the practical application of this treatment is that the hCPCs of elderly patients, who are most susceptible to myocardial disease, are senescent and prone to cell death. Rejuvenating hCPCs from elderly patients may help overcome this obstacle, and can be accomplished by reversing entry into the cellular stage of senescence. p16(INK4A), a cyclin dependent kinase inhibitor, is an important player in the regulation of cell senescence. In this study, we investigated whether knockdown of p16(INK4A) will rejuvenate aging hCPCs to a youthful phenotype. Our data indicated that upregulation of p16(INK4A) is associated with hCPC senescence. Both cell proliferation and survival capacity were significantly increased in hCPCs infected with lentivirus expressing p16(INK4A) shRNA when compared to control hCPCs. The knockdown of p16(INK4A) also induced antioxidant properties as indicated by a 50% decrease in ROS generation at basal cell metabolism, and a 25% decrease in ROS generation after exposure to oxidative stress. Genes associated with cell senescence (p21(CIP1)), anti-apoptosis (BCL2 and MCL1), anti-oxidant (CYGB, PRDX1 and SRXN1), and NFκB signal pathway (p65, IKBKB, HMOX1, etc.), were significantly upregulated after the p16(INK4A) knockdown. Knocking down the NFκB-p65 expression also significantly diminished the cytoprotective effect caused by the p16(INK4A) knockdown. Our results suggest that genetic knockdown of p16(INK4A) may play a significant role in inducing antioxidant effects and extending lifespan of aging hCPCs. This genetic modification may enhance the effectiveness of autologous hCPC therapy for repair of infarcted myocardium.

Ischemic heart disease affects a majority of people, especially elderly patients. Recent studies have utilized autologous adult stem/progenitor cells as a treatment option to heal cardiac tissue after myocardial infarction. However, donor cells from aging patients are more likely to be in a senescent stage. Rejuvenation is required to reverse the damage levied by aging and promote a youthful phenotype. This review aims to discuss current strategies that are effective in rejuvenating aging cardiac stem cells and represent novel therapeutic methods to treat the aging heart. Recent literature mainly focuses on three approaches that aim to reverse cardiac aging: genetic modification, pharmaceutical administration, and optimization of extracellular factors. In vitro genetic modification can be used to overexpress or knock down certain genes and allow for reversal of the aging phenotype. Pharmaceutical administration is another approach that allows for manipulation of signaling pathways related to cell proliferation and cell senescence. Since the stem cell niche can contribute to the age-related decline in stem cell function, rejuvenation strategies also include optimization of extracellular factors. Overall, improving the intrinsic properties of aging stem cells as well as the surrounding environment allows these cells to adopt a phenotype similar to their younger counterparts.

The regenerative potential of c-kit+ cardiac stem cells (CSCs) is severely limited by the poor survival of cells after transplantation in the infarcted heart. We have previously demonstrated that preconditioning human CSCs (hCSCs) with the heme oxygenase-1 inducer, cobalt protoporphyrin (CoPP), has significant cytoprotective effects in vitro. Here, we examined whether preconditioning hCSCs with CoPP enhances CSC survival and improves cardiac function after transplantation in a model of myocardial infarction induced by a 45-minute coronary occlusion and 35-day reperfusion in immunodeficient mice. At 30 minutes of reperfusion, CoPP-preconditioned hCSCsGFP+, hCSCsGFP+, or medium were injected into the border zone. Quantitative analysis with real-time qPCR for the expression of the human-specific gene HLA revealed that the number of survived hCSCs was significantly greater in the preconditioned-hCSC group at 24 hours and 7 and 35 days compared with the hCSC group. Coimmunostaining of tissue sections for both green fluorescent protein (GFP) and human nuclear antigen further confirmed greater hCSC numbers at 35 days in the preconditioned-hCSC group. At 35 days, compared with the hCSC group, the preconditioned-hCSC group exhibited increased positive and negative left ventricular (LV) dP/dt, end-systolic elastance, and anterior wall/apical strain rate (although ejection fraction was similar), reduced LV remodeling, and increased proliferation of transplanted cells and of cells apparently committed to cardiac lineage. In conclusion, CoPP-preconditioning of hCSCs enhances their survival and/or proliferation, promotes greater proliferation of cells expressing cardiac markers, and results in greater improvement in LV remodeling and in indices of cardiac function after infarction. Stem Cells 2015;33:3596–3607

Cardiac stem cells may serve in regenerative medicine to repair the infarcted heart. However, this approach is severely limited by the poor survival of donor cells. We previously showed that preconditioning human cardiac stem cells (hCSCs) with a nitric oxide (NO) donor enhanced cell survival through activation of survival signaling pathways. Recent studies suggest that the mammalian globin cytoglobin (CYGB) regulates NO metabolism and may regulate cell death. Therefore, the goal of the present study was to test the hypothesis that CYGB may serve important pro-survival functions in hCSCs. We found that CYGB is expressed in hCSCs, in the absence of any significant expression of myoglobin (MB), the primary hemoglobin expressed in adult cardiomyocytes. Through molecular approaches aimed at increasing or decreasing CYGB expression in hCSCs, we found that CYGB functions as a pro-survival factor in response to oxidative stress. This was associated with the upregulation of primary antioxidant systems such as peroxiredoxin 1 (PRDX1) and heme oxygenase-1 and anti-apoptotic factors, including BCL2, BCL-XL, and MCL1. Most significantly, we established that CYGB increased the expression of NFкB-dependent genes including iNOS (NOS2) and that iNOS-dependent NO production was required for a feedforward loop that maintains CYGB expression. Our study delineates for the first time a role for a globin in regulating hCSC survival and establishes novel mechanistic insights in the functional role of CYGB. It provides a rationale for the exploration of the CYGB pathway as a novel molecular target that can be used to enhance the effectiveness of stem cell therapy for ischemic heart disease.