Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Abstract 238: S-nitrosoglutathione Reductase (GSNOR) Plays a Critical Role in Placental Vascularization Working Through the VEGF-NO Pathway

Introduction: Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc), which is required for the release of nitric oxide (NO) from nitrosylated proteins. Mice lacking S-nitrosoglutathione reductase (GSNOR –/– ), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesized that impaired placental vascularization, one of the primary causes of preeclampsia is mediated by alteration in S-nitrosylation of the VEGF-NO pathway, and ascorbate treatment rescues this pathologic phenotype. Methods: Pregnant GSNOR –/– and control (WT) mice (n=5-7) were studied at late pregnancy (day 17.5). Ascorbate was provided in drinking water beginning at day 0.5. Fetoplacental capillary density was determined from isolectin staining and reactive nitrosative stress determined from nitrotyrosine staining in placental sections. S-nitrosylation of VEGF was determined using SNO-Rac and eNOS levels by Western blot analysis. Results: Fetoplacental capillary density was reduced 19% in GSNOR –/– fetuses compared to WT (P<0.001). GSNOR –/– placentas exhibited higher nitrotyrosine staining than WT placentas, indicating the presence of nitrosative stress. These increases were associated with reduced level of eNOS protein (P<0.05) and decreased S-nitrosylation of VEGF (P<0.05) in the GSNOR –/– placentas as compared to WT. Ascorbate treatment decreased nitrotyrosine staining, and increased fetoplacental capillary density ~10% (P<0.001), eNOS protein levels (P<0.05) and S-nitrosylation of VEGF (P<0.05) in the GSNOR –/– placentas as compared to WT. Conclusion: These findings suggest that GSNOR plays an essential role in promoting placental vascularization in part working through the VEGF-NO pathway. Ascorbate treatment rescued the nitrosative stress and improved placental vascularization, suggesting that it can be used therapeutically to treat or prevent preeclampsia

Abstract 215: Allogeneic MSCs Improve Endothelial Function in Patients with Dilated Cardiomyopathy via an SDF-1α-mediated Mechanism and the Suppression of Pathologic Cytokines

Endothelial dysfunction is central to the pathophysiology of heart failure, including dilated cardiomyopathy (DCM). Current drug therapies are unable to halt the progression of DCM, compelling the emergence of novel stem cell therapy approaches. Mesenchymal stem cells (MSCs) are pro-angiogenic, immunomodulatory, antifibrotic, and stimulate endogenous endothelial progenitor (EPC) proliferation and function, thus having the potential to ameliorate endothelial dysfunction. We demonstrated that patients with DCM who received allogeneic MSCs had a significant improvement in endothelial function 3-months post treatment, whereas patients who received autologous MSCs had no improvement. Therefore, we hypothesized that allogeneic MSCs preferentially improve endothelial function via a mechanism involving the suppression of pathologic levels of vascular endothelial growth factor (VEGF), stromal derived factor-1 alpha (SDF-1α), and tumor necrosis factor alpha (TNFα). Accordingly, patient serum VEGF and TNFα were measured at baseline and 3 months post MSC treatment. In vitro, MSC secretion of SDF-1α and TNFα was also measured. Our results show that patients with DCM had elevated levels of VEGF (n=21, 581.2±812.2 pg/mL) and TNFα (n=15, 22±9.4 pg/mL) at baseline, and that only allogeneic MSCs were able to restore these levels toward normal (VEGF: n=10, Δ-267.1±252.1, P=0.01; TNFα: n=8, Δ-7.1±3.1 pg/mL, P=0.0005). While there was no difference in TNFα secretion by autologous or allogeneic MSCs (0.01±0.14 vs. 0.4±0.6 pg/mL), autologous MSCs secreted significantly higher levels of SDF-1α compared to allogeneic MSCs (n=12, 79.3±16.7 vs. 14.2±9.4 pg/mL, P=0.0001). In vitro secreted SDF-1α and serum VEGF and TNFα levels correlated with EPC bioactivity (ΔSDF-1α to ΔEPC-CFUs, R=-0.9, P<0.0001; ΔVEGF to ΔEPC-CFUs, R=-0.7, P=0.001; ΔTNFα to ΔEPC-CFUs, R=-0.6, P=0.01). These findings reveal a novel mechanism by which allogeneic MSCs secrete physiologic levels of SDF-1α resulting in physiologic levels of VEGF signaling, reduced TNFα, increased EPC bioactivity, and improved endothelial function. These findings have important clinical and biological implications for the use of MSCs in patients with DCM

Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair

Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear. We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry. Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4 hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4 cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit cardiac cells. Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4 cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications

Acellular Human Amniotic Fluid-Derived Extracellular Vesicles as Novel Anti-Inflammatory Therapeutics against SARS-CoV-2 Infection

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is associated with acute respiratory distress syndrome (ARDS) and fatal pneumonia. Excessive inflammation caused by SARS-CoV-2 is the key driver of ARDS and lethal disease. Several FDA-approved drugs that suppress virus replication are in clinical use. However, despite strong evidence for the role of virus-induced inflammation in severe COVID-19, no effective anti-inflammatory drug is available to control fatal inflammation as well as efficiently clear the virus. Therefore, there is an urgent need to identify biologically derived immunomodulators that suppress inflammation and promote antiviral immunity. In this study, we evaluated acellular human amniotic fluid (acAF) containing extracellular vesicles (hAF-EVs) as a potential non-toxic and safe biologic for immunomodulation during COVID-19. Our in vitro results showed that acAF significantly reduced inflammatory cytokine production in TLR2/4/7 and SARS-CoV-2 structural protein-stimulated mouse macrophages. Importantly, an intraperitoneal administration of acAF reduced morbidity and mortality in SARS-CoV-2-infected mice. A detailed examination of SARS-CoV-2-infected lungs revealed that the increased protection in acAF-treated mice was associated with reduced viral titers and levels of inflammatory myeloid cell infiltration. Collectively, our results identify a novel biologic that has potential to suppress excessive inflammation and enhance survival following SARS-CoV-2 infection, highlighting the translational potential of acAF against COVID-19

Abstract P025: Modulatory Effect of Angiotensin II on Endothelial Differentiation and Vasculogenic Capacity of Human MSCs

Human bone marrow-derived mesenchymal stem cell (MSC)-based therapy holds great promise as a new approach for cardiovascular regeneration. In heart failure, there is activation of the renin-angiotensin system and increased formation of angiotensin (Ang) II. We tested the hypothesis that Ang II impairs endothelial differentiation and vasculogenic capacity of human MSCs. MSCs were cultured in endothelial growth media (EGM) and treated with vehicle, 1µM Ang II, or 10µM Ang II for 7 days to assess by real time RT-PCR the expression of two markers of endothelial differentiation, platelet endothelial cell adhesion molecule (PECAM) and von Willebrand factor (vWF). Expression of PECAM and vWF increased similarly in each treatment group at day 7, suggesting endothelial differentiation of MSCs. To assess vasculogenesis, MSCs were cultured in EGM for 7 days and then plated on growth factor reduced Matrigel for 5 hours under the following conditions: vehicle, Ang II (1µM), Ang II + type 1 receptor (AT1R) inhibitor (10µM losartan), or Ang II + type 2 receptor (AT2R) inhibitor (0.1µM PD123177). The vascular index (VI) was determined by multiplying the average number of endothelial tubes formed by the average tube length. MSCs treated for 5 hours with 1µM Ang II exhibited a reduced VI (84.5 ± 2.6% of vehicle treated cells, P<0.05). Treatment with Ang II+AT1R inhibitor increased VI (115 ± 4.9% of vehicle, P<0.05) whereas Ang II+AT2R inhibitor decreased VI (77.1 ± 4.1% of vehicle, P<0.05). Chronic treatment for 7 days in EGM with 1µM or 10µM Ang II also showed decreased VI compared to vehicle (75.4 ± 5.4% and 60.7 ± 2% of vehicle, respectively, P<0.01). These results reveal that both acute and chronic treatment with Ang II produces a negative effect on the vasculogenic potential of endothelial-differentiated human MSCs and suggests opposing effects of AT1R and AT2R on vasculogenesis. In conclusion, we propose that selective modulation of AT1R and AT2R mediated pathways may serve as a promising means for enhancing the regenerative capacity of human MSCs in cardiovascular disease

Systemic delivery of large-scale manufactured Wharton’s Jelly mesenchymal stem cell-derived extracellular vesicles improves cardiac function after myocardial infarction

Introduction: Cardiovascular disease and myocardial infarction are leading causes of morbidity and mortality in aged populations. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are under evaluation as a therapeutic option for the treatment of myocardial infarction.Aim: This study aimed to develop a large-scale manufacturing procedure to harvest clinical-grade EVs required for the translation of EVs to the clinic.Methods and Results: We compared the efficiency of large scale MSC-derived EV production and characterized EV miRNA cargo using the Quantum bioreactor with either fetal bovine serum or human platelet lysate (PLT)-containing expansion media. We tested the potency of the EV products in a murine model of acute myocardial infarction. Our results demonstrate an advantage of the Quantum bioreactor as a large-scale platform for EV production using PLT media; however, both media produced EVs with similar effects in vivo. The systemic delivery of EV products improved cardiac function following myocardial infarctions as indicated by a significant improvement in ejection fraction as well as parameters of cardiac performance, afterload, contractility and lusitropy.Conclusion: These findings have important implications for scale-up strategies of EVs and will facilitate clinical trials for their clinical evaluation.One sentence summary: Large scale manufacturing of MSC-derived EVs is feasible and when delivered systemically, improves cardiac function after myocardial infarction