Restoring the infected powerhouse: Mitochondrial quality control in sepsis.

Sepsis is a dysregulated host response to an infection, characterized by organ failure. The pathophysiology is complex and incompletely understood, but mitochondria appear to play a key role in the cascade of events that culminate in multiple organ failure and potentially death. In shaping immune responses, mitochondria fulfil dual roles: they not only supply energy and metabolic intermediates crucial for immune cell activation and function but also influence inflammatory and cell death pathways. Importantly, mitochondrial dysfunction has a dual impact, compromising both immune system efficiency and the metabolic stability of end organs. Dysfunctional mitochondria contribute to the development of a hyperinflammatory state and loss of cellular homeostasis, resulting in poor clinical outcomes. Already in early sepsis, signs of mitochondrial dysfunction are apparent and consequently, strategies to optimize mitochondrial function in sepsis should not only prevent the occurrence of mitochondrial dysfunction, but also cover the repair of the sustained mitochondrial damage. Here, we discuss mitochondrial quality control (mtQC) in the pathogenesis of sepsis and exemplify how mtQC could serve as therapeutic target to overcome mitochondrial dysfunction. Hence, replacing or repairing dysfunctional mitochondria may contribute to the recovery of organ function in sepsis. Mitochondrial biogenesis is a process that results in the formation of new mitochondria and is critical for maintaining a pool of healthy mitochondria. However, exacerbated biogenesis during early sepsis can result in accumulation of structurally aberrant mitochondria that fail to restore bioenergetics, produce excess reactive oxygen species (ROS) and exacerbate the disease course. Conversely, enhancing mitophagy can protect against organ damage by limiting the release of mitochondrial-derived damage-associated molecules (DAMPs). Furthermore, promoting mitophagy may facilitate the growth of healthy mitochondria by blocking the replication of damaged mitochondria and allow for post sepsis organ recovery through enabling mitophagy-coupled biogenesis. The remaining healthy mitochondria may provide an undamaged scaffold to reproduce functional mitochondria. However, the kinetics of mtQC in sepsis, specifically mitophagy, and the optimal timing for intervention remain poorly understood. This review emphasizes the importance of integrating mitophagy induction with mtQC mechanisms to prevent undesired effects associated with solely the induction of mitochondrial biogenesis.

Towards prevention of ischemia-reperfusion kidney injury: Pre-clinical evaluation of 6-chromanol derivatives and the lead compound SUL-138✰.

Acute kidney injury (AKI) is a global healthcare burden attributable to high mortality and staggering costs of dialysis. The underlying causes of AKI include hypothermia and rewarming (H/R), ischemia/reperfusion (I/R), mitochondrial dysfunction and reactive oxygen species production. Inspired by the mechanisms conferring organ protection in hibernating hamster, 6-chromanol derived compounds were developed to address the need of effective prevention and treatment of AKI. Here we report on the pre-clinical screening of 6-chromanol leads that confer protection during I/R to select compounds with favorable profiles for clinical testing in AKI. A library of 6-chromanols (n = 63) was screened in silico for pharmacochemical properties and druggability. Selected compounds (n = 15) were screened for the potency to protect HEK293 cells from H/R cell death and subjected to a panel of in vitro safety assays. Based on these parameters, SUL-138 was selected as the lead compound and was found to safeguard kidney function and decrease renal injury after I/R in rats. The compound was without cardiovascular or respiratory effects in vivo. SUL-138 pharmacokinetics of control animals (mouse, rat) and those undergoing I/R (rat) was identical, showing a two-phase elimination profile with terminal half-life of about 8 h. Collectively, our phenotype-based screening approach led to the identification of 3 candidates for pre-clinical studies (5%, 3/64). SUL-138 emerged from this small-scale library of 6-chromanols as a novel prophylactic for AKI. The presented efficacy and safety data provide a basis for future development and clinical testing. SECTION ASSIGNMENTS: : Drug discovery and translational medicine, renal, metabolism SIGNIFICANCE STATEMENT: : Based on in silico druggability parameters, a 63 compound 6-chromanol library was narrowed down to 15 compounds. These compounds were subjected to phenotypical screening of cell survival following hypothermia damage and hit compounds were identified. After subsequent assessment of in vivo efficacy, toxicity, pharmacokinetics, and cardiovascular and respiratory safety, SUL-138 emerged as a lead compound that prevented kidney injury after ischemia/reperfusion and demonstrated a favorable pharmacokinetic profile unaffected by renal ischemia.

The (R)-enantiomer of the 6-chromanol derivate SUL-121 improves renal graft perfusion via antagonism of the α1-adrenoceptor.

SUL-compounds are protectants from cold-induced ischemia and mitochondrial dysfunction. We discovered that adding SUL-121 to renal grafts during warm machine reperfusion elicits a rapid improvement in perfusion parameters. Therefore, we investigate the molecular mechanisms of action in porcine intrarenal arteries (PIRA). Porcine kidneys were stored on ice overnight and perfusion parameters were recorded during treatment with SUL-compounds. Agonist-induced vasoconstriction was measured in isolated PIRA after pre-incubation with SUL-compounds. Receptor binding and calcium transients were assessed in α1-adrenoceptor (α1-AR) transgenic CHO cells. Molecular docking simulation was performed using Schrödinger software. Renal pressure during warm reperfusion was reduced by SUL-121 (-11.9 ± 2.50 mmHg) and its (R)-enantiomer SUL-150 (-13.2 ± 2.77 mmHg), but not by the (S)-enantiomer SUL-151 (-1.33 ± 1.26 mmHg). Additionally, SUL-150 improved renal flow (16.21 ± 1.71 mL/min to 21.94 ± 1.38 mL/min). SUL-121 and SUL-150 competitively inhibited PIRA contraction responses to phenylephrine, while other 6-chromanols were without effect. SUL-150 similarly inhibited phenylephrine-induced calcium influx and effectively displaced [7-Methoxy-3H]-prazosin in CHO cells. Docking simulation to the α1-AR revealed shared binding characteristics between prazosin and SUL-150. SUL-150 is a novel α1-AR antagonist with the potential to improve renal graft perfusion after hypothermic storage. In combination with previously reported protective effects, SUL-150 emerges as a novel protectant in organ transplantation.

Combined implantation of CD34+ and CD14+ cells increases neovascularization through amplified paracrine signalling.

Cell therapy strategies that use adult peripheral blood-derived CD34⁺ progenitor cells are hampered by low cell numbers and the infrequent cellular incorporation into the neovasculature. Hence, the use of CD34⁺ cells to treat ischaemic diseases is under debate. Interaction between CD34⁺ cells and CD14⁺ cells results in superior endothelial differentiation of CD14⁺ cells in vitro, indicating that cell therapy approaches utilizing both CD34⁺ and CD14⁺ cells may be advantageous in therapeutic neovascularization. Here, human CD34⁺ and CD14⁺ cells were isolated from adult peripheral blood and implanted subcutaneously into nude mice, using matrigel as the carrier. Combined implantation of human CD34⁺ and CD14⁺ cells resulted in superior neovascularization, compared to either cell type alone, albeit incorporation of human cells into the murine vasculature was not observed. Human CD34⁺ and CD14⁺ cells produced and secreted a pentad of pro-angiogenic mediators, such as HGF, MCP-1 and IL-8, bFGF and VEGFa in monoculture. The production and secretion of pro-angiogenic mediators by CD14⁺ cells was highly amplified upon incubation with conditioned medium from CD34⁺ cells. In vivo, neovascularization of matrigel implants did not rely on the endothelial differentiation and incorporation of CD34⁺ or CD14⁺ cells, but depended on the paracrine effects of IL-8, MCP-1, HGF, bFGF and VEGFa secreted by implanted cells. Administration of this growth factor/cytokine pentad using matrigel as a carrier results in cell recruitment and microvessel formation equal to progenitor cell-induced neovascularization. These data provide new insights on neovascularization by cell therapy and may contribute to new strategies for the treatment of ischaemic diseases.

CD34+ cells augment endothelial cell differentiation of CD14+ endothelial progenitor cells in vitro.

Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34(+) cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34(+) cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14(+) cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14(+) cell-derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34(+) and CD14(+) cells augments endothelial differentiation of the CD14(+) cells. In vitro, the influence of CD34(+) cells on the endothelial differentiation capacity of CD14(+) cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14(+)-derived cells adopted an endothelial cell phenotype, whereas in CD34(+)/CD14(+) co-cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34(+)/CD14(+) co-cultures compared to both monocultures. CD34-conditioned medium also increased endothelial differentiation of CD14(+) cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin-8 and monocyte chemoattractant protein-1 neutralizing antibodies. We show that co-culturing of CD34(+) and CD14(+) cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction.