What is mitochondrial respiration

Whole-mount three-dimensional imaging of embryonic vascular network Fig. Together, these data clearly indicate that cox10 KO in ECs results in impaired vascular development and embryonic lethality in mice. The generated cox10 -deficient cell line is hereafter referred to as cox10 KO. Purity of the isolated cells and efficient in vitro deletion of the cox10 gene were first verified Supplementary Fig. As expected, the oxygen consumption rate OCR was drastically reduced in cox10 -deficient ECs with a significant reduction in the calculated basal and spare respiratory capacities Fig.

Furthermore, the fraction of basal respiration that was being used to drive ATP production as well as the remaining basal respiration that was not coupled to ATP production proton leak were both significantly reduced Fig.

The reduced respiratory capacity of cox10 -deficient ECs was accompanied by a compensatory increase in glycolysis Fig. After addition of saturating amounts of glucose, the extracellular acidification rate ECAR was boosted to the maximum glycolytic capacity in cox10 KO ECs. Comparable levels of glycolysis are only observed in wild-type ECs in the presence of oligomycin, which effectively shuts down OxPhos thus driving the cells to use glycolysis to its maximum capacity Fig.

Depending on the availability of nutrients, and glucose in particular, only few tissues possess the ability to switch between glycolysis and OxPhos, to adapt their metabolism to the prevailing environmental conditions. Thus, under saturating amounts of glucose, OxPhos in cox10 -competent ECs was suppressed. Together, these findings indicate that the mitochondrial dysfunction in COX-deficient ECs may be alleviated by the compensatory utilisation of glucose as ECs obviously have the ability to flexibly adjust their energy utilisation according to nutrient availability.

Metabolic profiling revealed that COX deficiency resulted in an accumulation of TCA cycle intermediates such as fumarate, malate and succinate, and reduction of glycolytic intermediates, and, in particular, significantly decreased cellular ATP levels indicating that EC mitochondrial respiration considerably contributes to cellular energy production Fig. Drop from bottom to top: : 0. Nucleotides from left to right: 0. Surprisingly, under standard cell culture conditions with physiological glucose concentrations, cox10 -deficient ECs exhibited no obvious morphological phenotype.

We therefore examined if glucose availability had an impact on the viability of COX-deficient ECs by using three independent viability assays in vitro Fig. In contrast, cox10 KO ECs lacking OxPhos were highly susceptible to glucose deprivation and responded with a gradual decrease in viability Fig.

Notably, glucose deprivation induced cell death in COX-deficient ECs which could not be rescued by the pan-caspase inhibitor zVAD-fmk excluding apoptosis as the main driver of EC death in this experimental system Supplementary Fig.

Individual data points in a , c and e represent technical replicates within a representative experiment. Individual data points in i and j represent mean values of individual mice of the respective genotype. Log-rank test was used to determine differences between survival curves in g , exact p -value: 0. Specifically, 3D spheroid sprouting assays 24 and scratch wound assays showed that sprouting response and migration of cox10 KO ECs was significantly hampered which could not be rescued by supra-physiological glucose concentrations Fig.

Cox10 -deficiency also significantly reduced EC proliferation Supplementary Fig. Notably, the pre-treatment of cells with mitomycin C, which is commonly used to block cell proliferation in cell migration assays, did not cause a gross delay in wound closure in our experimental setup Supplementary Fig. This suggests that the reduced migratory capacity of cox10 KO ECs rather than their reduced proliferation underlies the EC dysfunction measured in 3D spheroid sprouting and scratch wound assays.

Together, these data indicate that ECs are dependent on OxPhos to execute important cellular functions including proliferation, migration and sprouting.

We have shown above that endothelial COX deficiency is associated with embryonic lethality. The resulting animals express membrane-targeted tdTomato mT prior to Cre-mediated recombination. Upon tamoxifen treatment, endothelial eGFP expression was detected indicating successful recombination in the vasculature of various organs of tamoxifen-treated mice Fig.

Histological quantification of blood vessels revealed no difference between cox10 ECKO and cox10 wt animals Supplementary Fig. Furthermore, cox10 deletion induced by tamoxifen treatment did not affect survival in adult mice for up to days Fig. These data indicate that in the quiescent vasculature of adult mice under homeostatic conditions, endothelial OxPhos is dispensable. Importantly, despite lacking an obvious pathological phenotype, we were unable to culture ECs isolated from adult cox10 ECKO animals.

We assume that this may be related to the incapacity of cox10 -deficient ECs to proliferate in culture as already shown in Supplementary Fig. To study the angiogenic capacity of ECs isolated from cox10 ECKO versus cox10 wt mice, we performed the quantitative three-dimensional ex vivo mouse aortic ring assay Aortic rings isolated from cox10 ECKO mice revealed a significant reduction in sprout formation and vascular branching Fig.

Notably, a previous report 3 indicated that the inhibition of mitochondrial OxPhos by using oligomycin in HUVECs does not interfere with the sprouting capacity of ECs, which is in contrast to our observation. However, a follow-up study came to the conclusion, that mitochondrial defects did contribute to EC proliferation and survival The discrepancy was explained by the use of different experimental setups and timing Thus, results obtained with chemical inhibitors in cell culture should be considered with special caution Quiescent ECs in the healthy adult are estimated to have a turnover rate of several months 29 whereas ECs involved in neoangiogenesis during wound healing or tumour angiogenesis drastically increase their proliferation rate 30 , 31 , To explore the role of OxPhos dysfunction in a physiological regenerative process, we first examined the role of EC-specific cox10 deletion in a model of excisional skin wound healing.

In this model, timely wound closure depends critically on efficient vascularisation of newly forming granulation tissue. In order to visualise Cre recombination in this disease model, mice were crossed to the above mentioned Cre reporter line R26mTmG.

Wound closure was delayed in EC-specific cox10 -deficient mice compared with floxed control animals as assessed by macroscopic and histological analysis Fig. Specifically, a reduction in the vascular density was observed without affecting the pericyte coverage of existing vessels while no pericytes were detectable in the avascular wounds Supplementary Fig. These data clearly demonstrate that endothelial OxPhos is required for neovascularisation during wound healing in adult animals.

Individual data points in d , g , h , k , l , n , o and q represent mean values of individual mice of the respective genotype. Exact p -value one sample t -test, two-tailed : c 0. Next, we examined the role of EC OxPhos in tumour growth and vascularisation in mice.

Both tumour models showed reduced tumour vascularisation while vascular support structures pericyte coverage where unaffected Fig. Tumour vessel disintegration has been shown to promote metastatic progression of tumours 33 , Accordingly, we also analysed lungs harvested from LLC s. Collectively, these data indicated that tumour ECs are dependent on functional OxPhos in order to form tumour vasculature, to supply nutrients and to support tumour growth and vascularisation.

In the tumour microenvironment glucose availability is limited due to the consumption of glucose by highly glycolytic tumour cells. As a result of glycolysis in the hypoxic tumour cells, pyruvate is formed and converted into lactate. It has been demonstrated that this may be utilised by oxygenated tumour cells to fuel OxPhos as a critical regulator of cancer development 35 , 36 , We hypothesised that lactate could also be metabolised by ECs in the tumour microenvironment, in a form of metabolic symbiosis with the tumour cells when glucose availability drops.

These findings indicate that tumour ECs are capable of metabolising lactate to fuel OxPhos. These observations highlight the propelling role of tumour-derived lactate for EC OxPhos-dependent neoangiogenesis. Individual data points in a , b , c , d and f represent technical replicates within a representative experiment.

Individual data points in h represent mean values of individual mice of the respective genotype. Conflicting observations have been made on the relative role of glycolysis versus mitochondrial respiration in ECs 3 , 8. Although it is increasingly being recognised that OxPhos is a player in EC metabolism, its significance has been debated This study seeks to directly address this controversy by genetically disrupting mitochondrial respiratory chain function in ECs in order to assess the relevance of OxPhos for EC function.

Our work now provides clear evidence that loss of OxPhos in ECs results in vascular dysfunction leading to i embryonic lethality, ii impairment of wound vascularisation with delayed wound healing and iii a reduction in tumour vascularisation and growth.

Our findings support previous studies showing that angiogenic ECs require both, an increase in glycolysis and OxPhos for the full EC angiogenic response 11 , Clearly the issue is complicated by the fact that different types of ECs cooperate during angiogenesis, each following a different metabolic programme with different energetic demands.

In this respect, two energetically different conditions may be considered; on the one hand, vascular maintenance with low endothelial energy consumption, and full abundance of metabolic substrates—on the other hand, EC sprouting with high energy demand during tumour growth occurring under challenging nutrient conditions. In the low energy demand situation of vascular maintenance, ECs meet their energetic requirements largely by glycolysis. Although less energy efficient than OxPhos, this has the advantage of reducing the production of potentially damaging reactive oxygen species.

In contrast, in the high energy demand situation, e. For cancer cells a symbiotic metabolic interaction has been described whereby cancer cells in hypoxic tumour areas metabolise glucose through anaerobic glycolysis whereas well-oxygenated cancer cells in the vicinity of blood vessels, consume lactate discarded by the hypoxic cancer cells to fuel mitochondrial metabolism 35 , Similar symbiotic metabolic relationships have also been observed in the brain, where under homeostatic conditions, lactate derived from glycolytic astrocytes serves as a metabolite for OxPhos in neurons 40 , In analogy, our data demonstrate that in the tumour vasculature equivalent interactions appear to operate.

Specifically, lactate derived from tumour cells can be metabolised by ECs of the tumour vasculature through OxPhos in order to cover the energetic demands under conditions of limited glucose availability.

Together our data show a much greater energetic dependency of the tumour vasculature on OxPhos than has previously been appreciated During the preparation of this manuscript, Diebold et al. While Diebold et al. Our data based on the EC specific disruption of COX in both, embryonic development and adult mice highlight the need for mitochondrial OxPhos for ATP production during angiogenic growth. These findings are supported by our previous demonstration that the energetic dependence of tumour blood vessels on OxPhos can be exploited therapeutically with mitochondrial uncoupling agents 8.

More recently, mitochondrial uncouplers have also been reported to inhibit tumour growth directly by antagonizing the anabolic effects of aerobic glycolysis The simultaneous targeting of both tumour cells and blood vessels by mitochondrial targeting agents could offer an advantage over conventional cancer therapies and should be explored further.

Tie2-Cre mice were received from Dirk Wohlleber Animal genotyping primers are summarized in Supplementary Table 1. For timed matings a single male was paired with a female mouse which was then plug checked daily. For embryonic staging the day with a positive plug check was counted as E0. Samples were incubated in primary rabbit anti-CD31 antibody , Abcam for 7 days, followed by intensive washing with PBSG-T and 2-day incubation in secondary Alexa Fluor goat anti-rabbit antibody , Life Technologies.

Tissue clearing was achieved by dehydration in ethanol and incubation in ECi until samples were translucent and subsequently imaged by confocal microscopy. Mice used for experiments were between 8 and 16 weeks old. In tumour or wound-healing experiments, the tumour injection or wounding was performed at day 3 after the last tamoxifen dose. Wound-healing experiments were performed as previously described 8 , Tamoxifen was given per oral gavage 5 days in a row, once daily until 3 days before wounding.

Wounding and preparation of wound tissue for histology was performed as described previously Louis, MO, USA , a glucose analog that inhibits the initial step of glycolysis by its interaction with hexokinase Fig. To determine glucose uptake levels, cells were incubated with a fluorescent D-glucose analog 2-[N- 7-nitobenzoxa-1,3-diazolyl -amino]deoxy-D glucose 2-NBDG; Fig.

OCR, a measure of oxygen utilization of cells, is an important indicator of mitochondrial function Extracellular acidification rate ECAR is a measure of lactic acid levels, formed during the conversion of glucose to lactate during glycolysis The following inhibitors were injected: oligomycin A 1. This allowed for calculation of OCR-linked ATP production, maximal respiration capacity and spare respiratory capacity.

Basal respiration was measured prior to addition of oligomycin A. This allowed for calculation of glycolysis rate, glycolytic capacity, and glycolytic reserve. Basal ECAR was measured prior to addition of glucose. After blocking with 2. Total sprout length and the number of sprouts were quantified of at least 10 spheroids for every condition.

All analyses were carried out in a blinded fashion. Quantification was carried out using Neuron-J plug-inn package for Image-J software The in ovo CAM assay was performed as described earlier 73 , On embryo developmental day EDD 3, a small hole was prepared in the eggshell and covered with parafilm Pechinery, Menasha, WI, USA to prevent dehydration and possible infections and eggs were returned to the incubator. The latter was analyzed and quantified manually by two independent observers and carried out in a blinded fashion.

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Such a difference could be due to compensatory adaptations in women in the oxygen cascade Della Torre and Maggi, Considering the greater life expectancy Seifarth et al. Future studies should focus on the possible in vivo physiological effects of a higher intrinsic mitochondrial respiration, identifying which mitochondrial components underlie a higher intrinsic mitochondrial respiration e. Additional factors such as diet, potential hormonal effects associated with the menstrual cycle are important questions for future study.

Whether the higher intrinsic mitochondrial function in women represents a compensatory peripheral adaptation to low blood oxygen content also remains an interesting question for related disciplines.

All authors contributed to the data collection. DC analyzed, interpreted the data, and wrote the first draft of the manuscript which was reviewed by FL and RB. All authors read and approved the final manuscript. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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