The synergism of cytosolic acidosis and reduced NAD+/NADH ratio is responsible for lactic acidosis-induced vascular smooth muscle cell impairment in sepsis.

BACKGROUND: During sepsis, serve vascular dysfunctions lead to life-threatening multiple organ failure, due to vascular smooth muscle cells (VSMC) impairments, resulting in vasoplegia, hypotension and hypoperfusion. In addition, septic patients have an altered cell metabolism that leads to lactic acidosis. Septic patients suffering from lactic acidosis have a high risk of mortality. In addition, septic survivors are at risk of secondary vascular disease. The underlying mechanisms of whether and how lactic acidosis leads to the changes in VSMCs is not well understood. The aim of this study was to comprehensively investigate the effect of lactic acidosis on VSMCs and additionally compare the effects with those induced by pure acidosis and sodium lactate. METHODS: Primary human aortic smooth muscle cells (HAoSMCs) were treated for 48 h with lactic acidosis (LA_pH 6.8), hydrochloric acid (HCl_pH 6.8), sodium lactate (Na+-lactate_pH 7.4) and the respective controls (ctrl._pH 7.4; hyperosmolarity control: mannitol_pH 7.4) and comparatively analyzed for changes in (i) transcriptome, (ii) energy metabolism, and (iii) phenotype. RESULTS: Both types of acidosis led to comparable and sustained intracellular acidification without affecting cell viability. RNA sequencing and detailed transcriptome analysis revealed more significant changes for lactic acidosis than for hydrochloric acidosis, with lactate being almost ineffective, suggesting qualitative and quantitative synergism of acidosis and lactate. Bioinformatic predictions in energy metabolism and phenotype were confirmed experimentally. Lactic acidosis resulted in strong inhibition of glycolysis, glutaminolysis, and altered mitochondrial respiration which reduced cellular ATP content, likely due to increased TXNIP expression and altered NAD+/NADH ratio. Hydrochloric acidosis induced significantly smaller effects without changing the NAD+/NADH ratio, with the ATP content remaining constant. These metabolic changes led to osteo-/chondrogenic/senescent transdifferentiation of VSMCs, with the effect being more pronounced in lactic acidosis than in pure acidosis. CONCLUSIONS: Overall, lactic acidosis exerted a much stronger effect on energy metabolism than pure acidosis, whereas lactate had almost no effect, reflecting the qualitative and quantitative synergism of acidosis and lactate. As a consequence, lactic acidosis may lead to acute functional impairments of VSMC, sustained perturbations of the transcriptome and cellular dedifferentiation. Moreover, these effects may contribute to the acute and prolonged vascular pathomechanisms in septic patients.

Hypothesis-generating analysis of the impact of non-damaging metabolic acidosis on the transcriptome of different cell types: Integrated stress response (ISR) modulation as general transcriptomic reaction to non-respiratory acidic stress?

Extracellular pH is an important parameter influencing cell function and fate. Microenvironmental acidosis accompanies different pathological situations, including inflammation, hypoxia and ischemia. Research focussed mainly on acidification of the tumour micromilieu and the possible consequences on proliferation, migration and drug resistance. Much less is known regarding the impact of microenvironmental acidosis on the transcriptome of non-tumour cells, which are exposed to local acidosis during inflammation, hypoxia, ischemia or metabolic derailment. In the present hypothesis-generating study, we investigated the transcriptional impact of extracellular acidosis on five non-tumour cell types of human and rat origin, combining RNA-Sequencing and extensive bioinformatics analyses. For this purpose, cell type-dependent acidosis resiliences and acidosis-induced transcriptional changes within these resilience ranges were determined, using 56 biological samples. The RNA-Sequencing results were used for dual differential-expression analysis (DESeq and edgeR) and, after appropriate homology mapping, Gene Ontology enrichment analysis (g:Profiler), Ingenuity Pathway Analysis (IPA®), as well as functional enrichment analysis for predicted upstream regulators, were performed. Extracellular acidosis led to substantial, yet different, quantitative transcriptional alterations in all five cell types. Our results identify the regulator of the transcriptional activity NCOA5 as the only general acidosis-responsive gene. Although we observed a species- and cell type-dominated response regarding gene expression regulation, Gene Ontology enrichment analysis and upstream regulator analysis predicted a general acidosis response pattern. Indeed, they suggested the regulation of four general acidosis-responsive cellular networks, which comprised the integrated stress response (ISR), TGF-ß signalling, NFE2L2 and TP53. Future studies will have to extend the results of our bioinformatics analyses to cell biological and cell physiological validation experiments, in order to test the refined working hypothesis here.