MSC exosomes attenuate sterile inflammation and necroptosis associated with TAK1-pJNK-NFKB mediated cardiomyopathy in diabetic ApoE KO mice.

Introduction: Diabetes is a debilitating disease that leads to complications like cardiac dysfunction and heart failure. In this study, we investigated the pathophysiology of diabetes-induced cardiac dysfunction in mice with dyslipidemia. We hypothesize diabetes in ApoE knockout (ApoE-/-) mice induces cardiac dysfunction by increasing inflammation and necroptosis. Methods: ApoE-/- mice were divided into experimental groups: Control, Streptozotocin (STZ), STZ + MSC-Exo (mesenchymal stem cell-derived exosomes), and STZ+MEF-Exo (Mouse embryonic fibroblast derived exosomes). At Day 42, we assessed cardiac function, collected blood and heart tissues. Heart tissue samples were analyzed for inflammation, necroptosis, signaling mechanism, hypertrophy and adverse structural remodeling using histology, immunohistochemistry, western blotting, RT-PCR, cytokine array and TF array. Results and Discussion: STZ treated ApoE-/- mice developed diabetes, with significantly (p<0.05) increased blood glucose and body weight loss. These mice developed cardiac dysfunction with significantly (p<0.05) increased left ventricular internal diameter end diastole and end systole, and decreased ejection fraction, and fractional shortening. We found significant (p<0.05) increased expression of inflammatory cytokines TNF- a, IL-6, IL-1a, IL-33 and decreased IL-10 expression. Diabetic mice also exhibited significantly (p<0.05) increased necroptosis marker expression and infiltration of inflammatory monocytes and macrophages. MSC-Exos treated mice showed recovery of diabetes associated pathologies with significantly reduced blood glucose, recovered body weight, increased IL-10 secretion and M2 polarized macrophages in the heart. These mice showed reduced TAK1-pJNK-NFKB inflammation associated expression and improved cardiac function with significantly reduced cardiac hypertrophy and fibrosis compared to diabetic mice. Treatment with MEF-Exos did not play a significant role in attenuating diabetes-induced cardiomyopathy as these treatment mice presented with cardiac dysfunction and underlying pathologies observed in STZ mice. Conclusion: Thus, we conclude that cardiac dysfunction develops in diabetic ApoE-/- mice, arising from inflammation, necroptosis, and adverse tissue remodeling, which is ameliorated by MSC-Exos, a potential therapeutic for diabetes-induced cardiomyopathy.

Mesenchymal Stem Cell-Derived Exosomes Ameliorate Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (DOX) is an incessantly used chemotherapeutic drug that can cause detrimental dose-dependent effects such as cardiotoxicity and congestive heart failure. Hence, there is a need to discover innovative therapeutic approaches to counteract DOX-induced cardiotoxicity (DIC). MSC-Exos have shown to reduce apoptosis and cardiac fibrosis and promote cardiomyocyte proliferation in myocardial infracted mice. However, the effect of MSC-Exos on ameliorating DOX-induced pyroptosis has not been investigated. In this current study, H9c2 were first exposed to DOX to stimulate pyroptosis, followed by subsequent treatment with MSC-Exos, with further analysis performed through immunocytochemistry, western blotting, and RT-PCR. Our data depicted that post-treatment with MSC-Exos significantly (p < 0.05) reduced the HMGB1/TLR4 axis, inflammasome formation (NLRP3), pyroptotic markers (caspase-1, IL-1ß, and IL-18), and the pyroptotic executioner (GSDMD) in DOX-treated H9c2 cells. In conclusion, our data show that MSC-Exos attenuates inflammation-induced pyroptosis in our in vitro DIC model. Our findings indicate that MSC-Exos may serve as a promising therapeutic intervention for mitigating DIC, as they maintain the therapeutic capabilities of MSCs while circumventing the drawbacks associated with traditional stem cell therapy.

Role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in diabetic complications.

Cardiovascular disease (CVD) complications have remained a major cause of death among patients with diabetes. Hence, there is a need for effective therapeutics against diabetes-induced CVD complications. Since its discovery, proprotein convertase subtilisin/kexin type 9 (PCSK9) has been reported to be involved in the pathology of various CVDs, with studies showing a positive association between plasma levels of PCSK9, hyperglycemia, and dyslipidemia. PCSK9 regulates lipid homeostasis by interacting with low-density lipoprotein receptors (LDLRs) present in hepatocytes and subsequently induces LDLR degradation via receptor-mediated endocytosis, thereby reducing LDL uptake from circulation. In addition, PCSK9 also induces pro-inflammatory cytokine expression and apoptotic cell death in diabetic-CVD. Furthermore, therapies designed to inhibit PCSK9 effectively reduces diabetic dyslipidemia with clinical studies reporting reduced cardiovascular events in patients with diabetes and no significant adverse effect on glycemic controls. In this review, we discuss the role of PCSK9 in the pathogenesis of diabetes-induced CVD and the potential mechanisms by which PCSK9 inhibition reduces cardiovascular events in diabetic patients.

Immune interactions in pembrolizumab (PD-1 inhibitor) cancer therapy and cardiovascular complications.

The use of immunotherapies like pembrolizumab (PEM) is increasingly common for the management of numerous cancer types. The use of PEM to bolster T-cell response against tumor growth is well documented. However, the interactions PEM has on other immune cells to facilitate tumor regression and clearance is unknown and warrants further investigation. In this review, we present literature findings that have reported the interactions of PEM in stimulating innate and adaptive immune cells, which enhance cytotoxic phenotypes. This triggers secretion of cytokines and chemokines, which have both beneficial and detrimental effects. We also describe how this leads to the development of rare but underreported occurrence of PEM-induced immune-related cardiovascular complications that arise suddenly and progress rapidly to debilitating and fatal consequences. This review encourages further research and investigation of PEM-induced cardiovascular complications and other immune cell interactions in patients with cancer. As PEM therapy in treating cancer types is expanding, we expect that this review will inform health care professionals of diverse specializations of medicine like dermatology (melanoma skin cancers), ophthalmology (eye cancers), and pathology (hematological malignancies) about PEM-induced cardiac complications.

PTEN-AKT pathway attenuates apoptosis and adverse remodeling in ponatinib-induced skeletal muscle toxicity following BMP-7 treatment.

Tyrosine kinase inhibitors (TKIs) including ponatinib are commonly used to treat cancer patients. Unfortunately, TKIs induce cardiac as well as skeletal muscle dysfunction as a side effect. Therefore, detailed mechanistic studies are required to understand its pathogenesis and to develop a therapeutic treatment. The current study was undertaken to examine whether ponatinib induces apoptosis and apoptotic mechanisms both in vitro and in vivo models and furthermore to test the potential of bone morphogenetic protein 7 (BMP-7) as a possible treatment option for its attenuation. Sol8 cells, a mouse myogenic cell line was exposed to ponatinib to generate an apoptotic cell culture model and were subsequently treated with BMP-7 to understand its protective effects. For the in vivo model, C57BL/6J mice were administered with ponatinib to understand apoptosis, cell signaling apoptotic mechanisms, and adverse muscle remodeling and its attenuation with BMP-7. TUNEL staining, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) methods were used. Our data show significantly (p < 0.05) increased TUNEL staining, caspase-3, BAX/Bcl2 ratio in the in vitro model. Furthermore, our in vivo muscle data show ponatinib-induced muscle myopathy, and loss in muscle function. The observed muscle myopathy was associated with increased apoptosis, caspase-3 staining, and BAX/Bcl-2 ratio as confirmed with IHC and RT-PCR. Furthermore, our data show a significant (p < 0.05) increase in the involvement of cell signaling apoptotic regulator protein PTEN and a decrease in cell survival protein AKT. These results suggest that increased apoptosis following ponatinib treatment showed an increase in skeletal muscle remodeling, sarcopenia, and fibrosis. Furthermore, BMP-7 treatment significantly (p < 0.05) attenuated ponatinib-induced apoptosis, BAX/Bcl2 ratio, decreased PTEN, and increased cell survival protein AKT, decreased adverse muscle remodeling, and improved muscle function. Overall, we provide evidence that ponatinib-induces apoptosis leading to sarcopenia and muscle myopathy with decreased function which was attenuated by BMP-7.

BMP-7 Attenuates Sarcopenia and Adverse Muscle Remodeling in Diabetic Mice via Alleviation of Lipids, Inflammation, HMGB1, and Pyroptosis.

Diabetic myopathy involves hyperglycemia, oxidative stress, and inflammation. However, the role of hypercholesterolemia-induced inflammation-mediated pathological mechanisms leading to fibrosis, sarcopenia, deterioration of muscle, and muscle dysfunction in diabetes is not well understood. In this study, we investigated the novel role of bone morphogenetic protein-7 (BMP-7) in ameliorating metabolic alterations, inflammation, pyroptosis, TGF-ß/SMAD cell signaling mechanisms, and progression of diabetic myopathy. C57BL/6J mice were treated with saline, streptozotocin (STZ), or STZ+BMP-7. Diabetes was confirmed by increased fasting glucose levels and a glucose tolerance test. Gastrocnemius muscle and blood samples were collected for lipid and tissue analysis using various methods. A significant increase in hyperglycemia resulted in an increase in lipid accumulation, monocyte infiltration, and inflammation, as well as an increase in pyroptotic markers and signaling markers in diabetic muscle myocytes. A structural analysis showed significant muscle loss, and increased muscle deterioration and fibrosis leading to muscle dysfunction. BMP-7 attenuated pathological processes that resulted in significantly improved muscle function. We report, for the first time, that increased hyperlipidemia aggravates inflammation-induced pyroptosis, resulting in significant muscle loss, sarcopenia, and adverse skeletal muscle remodeling in diabetic muscle myopathy. Interventional treatment with BMP-7 attenuates hypercholesterolemia-induced inflammation-mediated sarcopenia and adverse muscle remodeling, suggesting BMP-7 could be a potential treatment option for diabetic muscle myopathy.

Overcoming the cardiac toxicities of cancer therapy immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have led recent advances in the field of cancer immunotherapy improving overall survival in multiple malignancies with abysmal prognoses prior to their introduction. The remarkable efficacy of ICIs is however limited by their potential for systemic and organ specific immune-related adverse events (irAEs), most of which present with mild to moderate symptoms that can resolve spontaneously, with discontinuation of therapy or glucocorticoid therapy. Cardiac irAEs however are potentially fatal. The understanding of autoimmune cardiotoxicity remains limited due to its rareness. In this paper, we provide an updated review of the literature on the pathologic mechanisms, diagnosis, and management of autoimmune cardiotoxicity resulting from ICIs and their combinations and provide perspective on potential strategies and ongoing research developments to prevent and mitigate their occurrence.

Mechanisms of COVID-19 pathogenesis in diabetes.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a global pandemic impacting 254 million people in 190 countries. Comorbidities, particularly cardiovascular disease, diabetes, and hypertension, increase the risk of infection and poor outcomes. SARS-CoV-2 enters host cells through the angiotensin-converting enzyme-2 receptor, generating inflammation and cytokine storm, often resulting in multiorgan failure. The mechanisms and effects of COVID-19 on patients with high-risk diabetes are not yet completely understood. In this review, we discuss the variety of coronaviruses, structure of SARS-CoV-2, mutations in SARS-CoV-2 spike proteins, receptors associated with viral host entry, and disease progression. Furthermore, we focus on possible mechanisms of SARS-CoV-2 in diabetes, leading to inflammation and heart failure. Finally, we discuss existing therapeutic approaches, unanswered questions, and future directions.

Inflammatory Cells in Atherosclerosis.

Atherosclerosis is a chronic progressive disease that involves damage to the intima, inflammatory cell recruitment and the accumulation of lipids followed by calcification and plaque rupture. Inflammation is considered a key mediator of many events during the development and progression of the disease. Various types of inflammatory cells are reported to be involved in atherosclerosis. In the present paper, we discuss the involved inflammatory cells, their characteristic and functional significance in the development and progression of atherosclerosis. The detailed understanding of the role of all these cells in disease progression at different stages sheds more light on the subject and provides valuable insights as to where and when therapy should be targeted.

Rat-Induced Pluripotent Stem Cells-Derived Cardiac Myocytes in a Cell Culture Dish.

Induced pluripotent stem (iPS) cells are genetically reprogrammed somatic cells that exhibit embryonic stem cell-like characteristics such as self-renewal and pluripotency. These cells have broad differentiation capability to convert into diverse cell types that make up the primary germ layers during embryonic development. iPS cells can spontaneously differentiate and form cell aggregates termed embryoid bodies (EBs) in the absence of differentiation inhibitory factors. Unlike other methods used to generate EBs, "the hanging drop" method offers reproducibility and homogeneity from a set number of iPS cells. As such, we describe the differentiation of rat-induced pluripotent stem cells into cardiac myocytes in vitro using the hanging drop method. Both the confirmation and identification of the cardiac myocytes are done using immunocytochemistry, RT-PCR, Western Blot, and Flow Cytometry. Briefly, a specific number of iPS cells are placed in droplets on the lid of culture dishes and incubated for 2 days, yielding embryoid bodies, which are suspended and plated. Spontaneous beating of cardiomyocytes can be seen 7-14 days after the plating of EBs and specific cardiac markers can be observed through identification assays.

Doxorubicin-induced apoptosis enhances monocyte infiltration and adverse cardiac remodeling in diabetic animals.

Diabetic cancer patients were treated with doxorubicin (DOX), a potent chemotherapeutic drug that induces cardiac toxicity; however, molecular mechanisms of cardiac toxicity in this specific disease progression in patients and animal models are completely unknown. Therefore, we designed a study to understand the effects of DOX-induced cardiac toxicity in diabetic animals and the involved pathophysiological mechanisms. C57BL/6 J mice were divided into four DOX- and diabetic (streptozotocin; STZ) - treated groups; control, STZ, DOX, and DOX+STZ. At day 14, animals were sacrificed, echocardiography was used to examine heart function, and heart and blood samples were collected to investigate apoptotic mechanisms (caspase 3, BAX, B-Cell leukemia/lymphoma 2 (Bcl2)), inflammation, and cardiac remodeling. Our data shows a significant (p < 0.05) increase in glucose levels, apoptotic markers, and monocyte infiltration at the site of apoptosis and triggered inflammatory immune response (tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)), in DOX+STZ animals compared with control and experimental groups. We also observed significant (p < 0.05) increase in myofibrillar area, fibrosis, and significantly decreased (p < 0.05) cardiac function in DOX-treated diabetic animals compared with controls. In conclusion, our data suggest that DOX induces significantly increased apoptosis, fibrosis, and structural alterations in diabetic hearts compared with non-diabetic animals.

BMP-7 Attenuates Inflammation-Induced Pyroptosis and Improves Cardiac Repair in Diabetic Cardiomyopathy.

In the present study, we investigated a novel signaling target in diabetic cardiomyopathy where inflammation induces caspase-1-dependent cell death, pyroptosis, involving Nek7-GBP5 activators to activate the NLRP3 inflammasome, destabilizes cardiac structure and neovascularization. Furthermore, we explored the therapeutic ability of bone morphogenetic protein-7 (BMP-7) to attenuate these adverse effects. C57BL/6J mice (n = 16 mice/group) were divided into: control (200 mg/kg, 0.9% saline intraperitoneal injection, i.p.); Streptozotocin (STZ) and STZ-BMP-7 groups (STZ, 200 mg/kg, i.p. injection). After 6 weeks, heart function was examined with echocardiography, and mice were sacrificed. Immunostaining, Western blotting, H&E, and Masson's trichrome staining was performed on heart tissues. STZ-induced diabetic cardiomyopathy significantly increased inflammasome formation (TLR4, NLRP3, Nek7, and GBP5), pyroptosis markers (caspase-1, IL-1ß, and IL-18), inflammatory cytokines (IL-6 and TNF-α), MMP9, and infiltration of monocytes (CD14), macrophage (iNOS), and dendritic cells (CD11b and CD11c) (p < 0.05). Moreover, a significant endothelial progenitor cells (EPCs) dysfunction (c-Kit/FLk-1, CD31), adverse cardiac remodeling, and reduction in left ventricular (LV) heart function were observed in STZ versus control (p < 0.05). Treatment with BMP-7 significantly reduced inflammasome formation, pyroptosis, and inflammatory cytokines and infiltrated inflammatory cells. In addition, BMP-7 treatment enhanced EPC markers and neovascularization and subsequently improved cardiac remodeling in a diabetic heart. Moreover, a significant improvement in LV heart function was achieved after BMP-7 administration relative to diabetic mice (p < 0.05). In conclusion, BMP-7 attenuated inflammation-induced pyroptosis, adverse cardiac remodeling, and improved heart function via the TLR4-NLRP3 inflammasome complex activated by novel signaling Nek7/GBP5. Our BMP-7 pre-clinical studies of mice could have significant potential as a future therapy for diabetic patients.

Role of phosphodiesterase 1 in the pathophysiology of diseases and potential therapeutic opportunities.

Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca2+/calmodulin (CaM), resulting in the integration of Ca2+ and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.

Amelioration of diabetes-induced inflammation mediated pyroptosis, sarcopenia, and adverse muscle remodelling by bone morphogenetic protein-7.

BACKGROUND: Diabetic myopathy involves hyperglycaemia and inflammation that causes skeletal muscle dysfunction; however, the potential cellular mechanisms that occur between hyperglycaemia and inflammation, which induces sarcopenia, and muscle dysfunction remain unknown. In this study, we investigated hyperglycaemia-induced inflammation mediating high-mobility group box 1 activation, which is involved in a novel form of cell death, pyroptosis, diabetic sarcopenia, atrophy, and adverse muscle remodelling. Furthermore, we investigated the therapeutic potential of bone morphogenetic protein-7 (BMP-7), an osteoporosis drug, to treat pyroptosis, and diabetic muscle myopathy. METHODS: C57BL6 mice were treated with saline (control), streptozotocin (STZ), or STZ + BMP-7 to generate diabetic muscle myopathy. Diabetes was established by determining the increased levels of glucose. Then, muscle function was examined, and animals were sacrificed. Gastrocnemius muscle or blood samples were analysed for inflammation, pyroptosis, weight loss, muscle atrophy, and adverse structural remodelling of gastrocnemius muscle using histology, enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and reverse transcription polymerase chain reaction. RESULTS: A significant (P < 0.05) increase in hyperglycaemia leads to an increase in inflammasome (high-mobility group box 1, toll-like receptor-4, and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing protein 3) formation in diabetic muscle cells. Further analysis showed an up-regulation of the downstream pyroptotic pathway with significant (P < 0.05) number of positive muscle cells expressing pyroptosis-specific markers [caspase-1, interleukin (IL)-1ß, IL-18, and gasdermin-D]. Pyroptotic cell death is involved in further increasing inflammation by releasing pro-inflammatory cytokine IL-6. Structural analysis showed the loss of muscle weight, decreased myofibrillar area, and increased fibrosis leading to muscle dysfunction. Consistent with this finding, BMP-7 attenuated hyperglycaemia (~50%), pyroptosis, inflammation, and diabetic adverse structural modifications as well as improved muscle function. CONCLUSIONS: In conclusion, we report for the first time that increased hyperglycaemia and inflammation involve cellular pyroptosis that induces significant muscle cell loss and adverse remodelling in diabetic myopathy. We also report that targeting pyroptosis with BMP-7 improves diabetic muscle pathophysiology and muscle function. These findings suggest that BMP-7 could be a potential therapeutic option to treat diabetic myopathy.

Stem Cell-Derived Exosomes Ameliorate Doxorubicin-Induced Muscle Toxicity through Counteracting Pyroptosis.

Doxorubicin (Dox)-induced muscle toxicity (DIMT) is a common occurrence in cancer patients; however, the cause of its development and progression is not established. We tested whether inflammation-triggered cell death, "pyroptosis" plays a role in DIMT. We also examined the potential role of exosomes derived from embryonic stem cells (ES-Exos) in attenuating DIMT. C57BL/6J mice (10 ± 2 wks age) underwent the following treatments: Control (saline), Dox, Dox+ES-Exos, and Dox+MEF-Exos (mouse-embryonic fibroblast-derived exosomes, negative control). Our results demonstrated that Dox significantly reduced muscle function in mice, which was associated with a significant increase in NLRP3 inflammasome and initiation marker TLR4 as compared with controls. Pyroptosis activator, ASC, was significantly increased compared to controls with an upregulation of specific markers (caspase-1, IL-1ß, and IL-18). Treatment with ES-Exos but not MEF-Exos showed a significant reduction in inflammasome and pyroptosis along with improved muscle function. Additionally, we detected a significant increase in pro-inflammatory cytokines (TNF-α and IL-6) and inflammatory M1 macrophages in Dox-treated animals. Treatment with ES-Exos decreased M1 macrophages and upregulated anti-inflammatory M2 macrophages. Furthermore, ES-Exos showed a significant reduction in muscular atrophy and fibrosis. In conclusion, these results suggest that DIMT is mediated through inflammation and pyroptosis, which is attenuated following treatment with ES-Exos.

The Role of Bone Morphogenetic Protein 7 (BMP-7) in Inflammation in Heart Diseases.

Bone morphogenetic protein-7 is (BMP-7) is a potent anti-inflammatory growth factor belonging to the Transforming Growth Factor Beta (TGF-ß) superfamily. It plays an important role in various biological processes, including embryogenesis, hematopoiesis, neurogenesis and skeletal morphogenesis. BMP-7 stimulates the target cells by binding to specific membrane-bound receptor BMPR 2 and transduces signals through mothers against decapentaplegic (Smads) and mitogen activated protein kinase (MAPK) pathways. To date, rhBMP-7 has been used clinically to induce the differentiation of mesenchymal stem cells bordering the bone fracture site into chondrocytes, osteoclasts, the formation of new bone via calcium deposition and to stimulate the repair of bone fracture. However, its use in cardiovascular diseases, such as atherosclerosis, myocardial infarction, and diabetic cardiomyopathy is currently being explored. More importantly, these cardiovascular diseases are associated with inflammation and infiltrated monocytes where BMP-7 has been demonstrated to be a key player in the differentiation of pro-inflammatory monocytes, or M1 macrophages, into anti-inflammatory M2 macrophages, which reduces developed cardiac dysfunction. Therefore, this review focuses on the molecular mechanisms of BMP-7 treatment in cardiovascular disease and its role as an anti-fibrotic, anti-apoptotic and anti-inflammatory growth factor, which emphasizes its potential therapeutic significance in heart diseases.

Exosome Treatment Enhances Anti-Inflammatory M2 Macrophages and Reduces Inflammation-Induced Pyroptosis in Doxorubicin-Induced Cardiomyopathy.

Doxorubicin (Dox) is an effective antineoplastic agent used to treat cancers, but its use is limited as Dox induces adverse cardiotoxic effects. Dox-induced cardiotoxicity (DIC) can lead to heart failure and death. There is no study that investigates whether embryonic stem cell-derived exosomes (ES-Exos) in DIC can attenuate inflammation-induced pyroptosis, pro-inflammatory M1 macrophages, inflammatory cell signaling, and adverse cardiac remodeling. For this purpose, we transplanted ES-Exos and compared with ES-cells (ESCs) to examine pyroptosis, inflammation, cell signaling, adverse cardiac remodeling, and their influence on DIC induced cardiac dysfunction. Therefore, we used C57BL/6J mice ages 10 ± 2 weeks and divided them into four groups (n = 6-8/group): Control, Dox, Dox + ESCs, and Dox + ES-Exos. Our data shows that the Dox treatment significantly increased expression of inflammasome markers (TLR4 and NLRP3), pyroptotic markers (caspase-1, IL1-ß, and IL-18), cell signaling proteins (MyD88, p-P38, and p-JNK), pro-inflammatory M1 macrophages, and TNF-α cytokine. This increased pyroptosis, inflammation, and cell signaling proteins were inhibited with ES-Exos or ESCs. Moreover, ES-Exos or ESCs increased M2 macrophages and anti-inflammatory cytokine, IL-10. Additionally, ES-Exos or ESCs treatment inhibited significantly cytoplasmic vacuolization, myofibril loss, hypertrophy, and improved heart function. In conclusion, for the first time we demonstrated that Dox-induced pyroptosis and cardiac remodeling are ameliorated by ES-Exos or ESCs.

Exosomes derived from cardiac parasympathetic ganglionic neurons inhibit apoptosis in hyperglycemic cardiomyoblasts.

Diabetic cardiomyopathy is known to involve two forms of cardiac cell death: apoptosis and necrosis. However, it remains unknown whether hyperglycemia-induced apoptosis in the H9c2 cell culture system is inhibited by parasympathetic ganglionic neurons (PGN) derived exosomes (exos). We isolated PGN and sympathetic ganglionic neurons (SGN) from the right stellate ganglion in rats, and derived exos from these sources. H9c2 cells were divided into 4 groups: (1) Control, (2) H9c2 + Glucose (100 mmol/L), (3) H9c2 + Glucose + PGN-exos, and (4) H9c2 + Glucose + SGN-exos. We determined cell proliferation and viability with an MTT assay kit, and assessed apoptotic cell death with TUNEL staining and ELISA. Data were further confirmed by analyzing the presence of pro-apoptotic proteins Caspase-3 and Bax, and anti-apoptotic protein Bcl-2. Glucose exposed H9c2 cells significantly reduced cell viability, which was improved by PGN-exos, but not by SGN-exos. Furthermore, increased apoptosis in hyperglycemia in H9c2 cells was confirmed with TUNEL staining and cell death ELISA which demonstrated significantly (p < 0.05) reduction with PGN-exos treatment, but not with SGN-exos. Moreover, high expression of pro-apoptotic proteins Caspase-3 and Bax was reduced following treatment with PGN-exos; however, SGN-exos were unable to reduce the expression. Significantly reduced anti-apoptotic protein Bcl-2 following glucose treatment was improved with PGN-exos. Therefore, our data suggest that hyperglycemia induces apoptosis in H9c2 cells and decreases cell viability, and that PGN-exos are able to inhibit apoptosis, improve cell viability, and restore levels of anti-apoptotic protein Bcl-2.

Embryonic stem cell-derived exosomes inhibit doxorubicin-induced TLR4-NLRP3-mediated cell death-pyroptosis.

Doxorubicin (Dox)-induced cardiac side effects are regulated through increased oxidative stress and apoptosis. However, it remains unknown whether Dox induces the specific inflammatory-mediated form of cell death called pyroptosis. The current study is undertaken to determine whether Dox induces pyroptosis in an in vitro model and to test the potential of exosomes derived from embryonic stem cells (ES-Exos) in inhibiting pyroptosis. H9c2 cells were exposed to Dox to generate pyroptosis and then subsequently treated with exosomes to investigate the protective effects of ES-Exos. Mouse embryonic fibroblast-exosomes (MEF-Exos) were used as a cell line control. We confirmed pyroptosis by analyzing the presence of Toll-like receptor 4 (TLR4)-pyrin domain containing-3 (NLRP3) inflammasome that initiates pyroptosis, which was further confirmed with pyroptotic markers caspase-1, IL-1ß, caspase-11, and gasdermin-D. The presence of inflammation was confirmed for proinflammatory cytokines, TNF-α, and IL-6. Our data show that Dox exposure significantly (P < 0.05) increases expression of TLR4, NLRP3, pyroptotic markers (caspase-1, IL-1ß, caspase-11, and gasdermin-D), and proinflammatory cytokines (TNF-α and IL-6) in H9c2 cells. The increased expression of inflammasome, pyroptosis, and inflammation was significantly (P < 0.05) inhibited by ES-Exos. Interestingly, our cell line control, MEF-Exos, did not show any protective effects. Furthermore, our cytokine array data suggest increased anti-inflammatory (IL-4, IL-9, and IL-13) and decreased proinflammatory cytokines (Fas ligand, IL-12, and TNF-α) in ES-Exos, suggesting that anti-inflammatory cytokines might be mediating the protective effects of ES-Exos. In conclusion, our data show that Dox induces pyroptotic cell death in the H9c2 cell culture model and is attenuated via treatment with ES-Exos.NEW & NOTEWORTHY Doxorubicin (Dox)-induced cardiotoxicity is mediated through increased oxidative stress, apoptosis, and necrosis. We report for the first time as per the best of our knowledge that Dox initiates Toll-like receptor 4 and pyrin domain containing-3 inflammasome formation and induces caspase-1-mediated inflammatory pyroptotic cell death in H9c2 cells. Moreover, we establish that inflammation and pyroptosis is inhibited by embryonic stem cell-derived exosomes that could be used as a future therapeutic option to treat Dox-induced cardiotoxicity.

Macrophage depletion by clodronate attenuates bone morphogenetic protein-7 induced M2 macrophage differentiation and improved systolic blood velocity in atherosclerosis.

Bone morphogenetic protein-7 (BMP-7) affects the presence of macrophage subtypes in vitro and in vivo at an early stage of atherosclerosis (ATH); however, it remains unknown whether BMP-7 treatment affects the development and progression of ATH at a mid-stage of the disease. We therefore performed a Day 28 (D28) study to examine BMP-7's potential to affect monocyte differentiation. Atherosclerosis was developed in ApoE KO mice, and these animals were treated with intravenous injections of BMP-7 and/or liposomal clodronate (LC). BMP-7 significantly (P < 0.05) lowers plaque formation following induction of atherosclerosis. However, upon macrophage depletion, BMP-7 fails to significantly alter plaque progression suggesting a direct role of BMP-7 on macrophages. Immunohistochemical staining of carotid arteries was performed to determine BMP-7's effect on pro-inflammatory M1 inducible nitric oxide synthase and anti-inflammatory M2 (cluster of differentiation [CD]206, Arginase-1) macrophages, and monocytes ( CD14). BMP-7 significantly reduced pro-inflammatory M1 macrophages and increased anti-inflammatory M2 macrophages at D28, while BMP-7 showed no effect on M2 macrophage differentiation in animals treated with LC. Enzyme-linked immunosorbent assay data showed significant reduction in proinflammatory cytokines (Interleukin-6 [IL-6]), monocyte chemoattractant protein-1, and tumor necrosis factor-α) and a significant increase in anti-inflammatory cytokine (IL-10) in BMP-7 treated mice (P < 0.05).Western blot analysis of arterial tissue confirms a significant increase in pro-survival kinases extracellular-signal regulated kinase and SMAD and a reduction in pro-inflammatory kinases p38 and c-Jun N-terminal kinase in BMP-7 treated mice (P < 0.05). Overall, this study suggests that clodronate treatment inhibits BMP-7 induced differentiation of monocytes into M2 macrophages and improved systolic blood velocity.