Where Metabolism Meets Senescence: Focus on Endothelial Cells

1Aging is the leading single risk factor for the development of most, if not all, major age-related chronic diseases, such as neurodegenerative, cancer, metabolic and cardiovascular diseases (CVDs). Aging and age-related diseases (ARDs) share a common set of basic biological mechanisms, such as inflammation, the accumulation of macromolecular damage, adaptation to molecular and psychological stressors, epigenetic changes, metabolic dysfunction, loss of proteostasis, and defective stem cell function. Major ARDs include type 2 diabetes (T2DM), cardiovascular diseases (CVDs), osteoporosis and certain types of cancers. The low-grade, chronic, and systemic inflammation underlying the aging process and ARDs was called “inflammaging”.

2The notion that the lifespan of many species can be extended through reduction of energy intake suggests a critical role of macronutrient metabolism in the control of regulatory processes influencing proliferation, survival, and ARD development. Accordingly, obesity is a risk factor for many ARDs and carries out a life-shortening action. The onset of ARDs can be counteracted through overweight reduction by decreasing the energetic food and by increasing energy expenditure with physical activity. Notably, centenarians, individuals capable of reaching the extreme limit of human life and characterized by an exceptionally healthy phenotype, share features observed in human adult volunteers who followed caloric restriction (CR) regimens. All these evidences suggest that major pathways driving organismal aging are intimately connected with metabolism, a hypothesis also confirmed by the fact that dysfunctional mitochondria are a common feature of aged cells and major ARDs.

3Within the cells, energy is generated in the form of adenosine triphosphate (ATP), mainly through mitochondrial oxidative phosphorylation (OXPHOS) in the presence of oxygen, and through anaerobic glycolysis in its absence. This “general rule” could be subjected to modifications during particular conditions, such as cellular senescence. Senescent cells (SCs) exhibit some peculiar characteristics, including growth arrest, telomere shortening, enhanced senescence associated (SA) β-Galactosidase activity, and the acquisition of a pro-inflammatory senescence-associated secretory phenotype (SASP), which is responsible for both inflammaging and the spreading of senescence to neighboring cells. Beyond the exhaustion of replicative potential, cellular senescence can also be triggered by different stressors, including hypoxia, endotoxin, and reactive oxygen species (ROS).

4Endothelial cells (ECs) form the inner lining of blood vessels. In adult organisms they are supposed to remain in a quiescent state, but they can be rapidly activated by a variety of stimuli. They provide a significant contribution to the transduction of signals between blood and tissues; moreover, by the release of the gaseous mediator nitric oxide (NO), ECs play a crucial role in maintaining the vascular tone and in preventing platelet aggregation. The integrity of the EC monolayer is a critical requisite to allow blood flow and avoid uncontrolled thrombosis. Growing evidence show that dysfunctional senescent ECs can play a key role in instigating the morphological and biochemical changes that accompany vascular dysfunction, thus not a mere epiphenomenon in pathogenesis of CVDs. For this reason, senescence of ECs has become a central focus of the investigations on ARDs.

5Here, we review the latest findings on the metabolic changes that occur during the aging process of endothelial cells. Moreover, due to EC strategic location in the human organism, here we support the hypothesis of a potential role of senescence-associated metabolic alterations of ECs in the onset and development of ARDs. Finally, the potential relevance of targeting specific EC metabolic features to counteract ARDs will be discussed.

6A peculiar feature of SCs is that they remain metabolically active, despite their growth arrest. Their high metabolic rate is intimately linked to SASP acquisition, but whether it is a cause or an effect of the inflammatory phenotype and altered proliferative status of SCs has yet to be established.

7Recent literature confers to metabolic reprogramming a deterministic role in modulating the inflammatory responses of the innate immune cells: in macrophages, metabolic pathways not only provide energy but also regulate their phenotype and function. Lipopolysaccharide (LPS)(+IFNγ)-activated proinflammatory (i.e., M1) macrophages mediate host defense through an enhanced glycolytic metabolism and impaired mitochondrial OXPHOS. On the contrary, interleukin (IL)-4(13)-activated (i.e., M2) macrophages, which promote wound healing and Th2-mediated responses, mainly rely on OXPHOS for the synthesis of ATP. These data suggest that metabolic changes may also underlie acquisition of the SASP in senescent cells.

8In the 1980s, the first attempts to analyze energy metabolism in aging cells showed that glucose consumption and lactate production are elevated in replicative senescent human diploid fibroblasts (HDFs). This “glycolytic state,” accompanied by an imbalance of the activity of the glycolytic enzymes, results in a less energetic state, mirrored by the drop of ATP and GTP intracellular levels as cell cultures enter in replicative senescence. More recently, metabolomic approaches on extracellular metabolites released by senescent fibroblasts confirmed a shift toward glycolysis compared to young cells. This general mechanism, however, is more or less pronounced depending on the cell type and shows some exceptions, such as senescent human mammary epithelial cells where glucose consumption and lactate secretion do not increase.

9The role of malic enzyme (ME) in senescent cells has been extensively investigated due to its role in maintaining cellular redox homeostasis. The two ME isoforms ME1 and ME2 catalyze the decarboxylation of malate to pyruvate with concomitant formation NADPH or NADH, respectively. Regeneration of reducing equivalents for anabolic processes in form of NADPH can be achieved either through the “malate oxidation shunt” or through the pentose phosphate pathway (PPP). In the context of aging cell, p53 by binding to specific response elements, attenuates ME1 and ME2 transcription. At the same time ME depletion induces senescence by stabilizing p53, suggesting that also ME exerts an inhibitory effect on p53. Moreover, expression of both malate dehydrogenase (MDH)1, a mitochondrial tricarboxylic acid (TCA) cycle enzyme catalyzing malate oxidation to oxaloacetate, and MDH2, the cytosolic enzyme of malate-aspartate shuttle, declines during aging, leading to an impaired transfer of reducing equivalents into the mitochondria. In HDF, the subsequent decrease of cytosolic NAD+/NADH ratio is accompanied by the induction of replicative senescence. The need to regenerate NAD+ explains the upregulation of lactate dehydrogenase (LDH) in senescent HDFs, which release lactate to avoid the arrest of glycolysis due to both product accumulation and excessively low intracellular pH (Figure 1). These observations on the role of malate prompted the hypothesis that malate supplementation could delay senescence and extend lifespan. Similar effects have been shown in C. elegans and D. melanogaster for α-ketoglutarate and oxaloacetate, two other TCA cycle intermediates. However, such observation needs to be corroborated by evidence in mammals.

10The role of PPP, the alternative source of NADPH, has been explored in cellular senescence. In fact, even if SCs have a lower demand of deoxyribonucleotides (dNTPs), they still require NADPH to allow the activity of the reactive oxygen species (ROS)-detoxifying thioredoxins, glutaredoxins and peroxiredoxins. Importantly, replicative senescence can be triggered by a lack of dNTPs due to reduced substrate availability. The activity of the first enzyme of the oxidative branch of PPP, namely glucose-6-phosphate dehydrogenase (G6PDH), is decreased during senescence. Accordingly, G6PDH-deficient cells exhibit accelerated oxidant-induced senescence, a process that can be partially rescued by telomerase ectopic expression. Notably, transgenic mice overexpressing G6PDH display extended lifespan through increased NADPH levels and knockdown of the tumor suppressor ataxia-telangiectasia mutated (ATM) gene restores glucose flux throughout the PPP and allows cells to overcome senescence.

11The findings on the metabolic features of replicative SCs have been only in part confirmed in oncogene-induced senescence (OIS). Seminal studies reported that cells undergoing OIS have high glycolysis rate, along with an elevated OXPHOS activity, when compared to proliferating cells. In line with these findings, both pyruvate kinase (PK) isoforms, i.e., PKM1 and PKM2, and pyruvate dehydrogenase (PDH), two enzymes with a key role in glycolysis and in the conversion of pyruvate to acetyl-CoA, are upregulated in SCs. This leads to an enhanced use of pyruvate for the TCA cycle causing increased cellular respiration and redox stress. The TCA cycle could be also fueled by metabolites from fatty acid catabolism. Indeed, Ras-induced senescent cells manifest a decline in lipid synthesis and an increase in fatty acid oxidation (FAO), which results in a higher rate of basal oxygen consumption. Inhibition of carnitine palmitoyltransferase 1A (CPT1A), the key rate-limiting enzyme for oxidation of free fatty acids (FFA) into the mitochondria, prevented senescence and SASP establishment. In a recent work, Fafián-Labora et al. demonstrated that fatty acid synthase (FASN) activity is important for mitochondrial bioenergetics in the initial phases of senescence. FASN is an enzyme that catalyzes de novo synthesis of fatty acids by combining malonyl-CoA to the acetyl-CoA derived from glycolysis-produced pyruvate. Indeed, inhibition of FASN activity prevented the p53-mediated induction of senescence, the secretion of the canonical SASP factors IL-1α, IL-1β, IL-6, and the release of extracellular vesicles (EVs) mediating the spread of pro-senescence signals at the paracrine level. Notably, other studies reported that p53 activation inhibits FASN, suggesting a negative feedback loop.

12High lipogenesis would explain the progressive accumulation of membranous organelles during cell senescence. It is well known that lysosome mass expands during senescence, recently an increase in mitochondrial mass was also established. A considerable number of senescent−associated changes are dependent on mitochondria. Indeed, mitochondrial dysfunction can elicit a specific type of proinflammatory phenotype, defined as mitochondrial dysfunction-associated senescence (MiDAS). MiDAS differs from the prototypical SASP for the lack of an IL-1/NF-κB-dependent mechanism. In MiDAS, a reduced NAD+/NADH ratio is believed to trigger adenosine monophosphate-activated protein kinase (AMPK) and p53 activation.

13NAD+/NADH ratio is one of the most reliable markers of the redox state of the cell. Its decrease has been reportedly linked to cellular senescence. NAD+ decline in aging is extensively reviewed by Verdin. Low levels of NAD+ were also reported in several tissues, and supplementation of NAD+ precursors increased life span in different species. Interestingly, SCs were shown to induce the SASP-mediated expression of CD38 – an ectoenzyme with a high NADase activity – in non-senescent cells, such as endothelial cells and bone marrow-derived macrophages. CD38 inhibitors rescued NAD+ decline and ameliorated a number of age-related metabolic outcomes in mice. A similar beneficial effect on mouse lifespan has been described also for the EV-mediated cell-to-cell transfer of the extracellular isoform of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD+ salvage pathway. EVs are membrane-coated nanoparticles actively released by almost all cell types, including ECs. EVs are usually categorized according to their size and surface markers, and are able to shuttle and deliver functional proteins and nucleic acids in a paracrine and systemic manner.

14Recently, demonstrated a direct link between intracellular NAD+ levels and the SASP in OIS. High SA-chromatin remodeling is related to the upregulation chromatin-binding protein High-Mobility Group A1 (HMGA1) which binds genomic A + T reach regions to increase chromatin accessibility. Interestingly, inhibition or silencing of NAMPT, one of the targets of HMGA1, decreased glycolysis, mitochondrial respiration, and oxygen consumption, along with NAD+/NADH ratio, which is normally elevated in OIS. The subsequent increase of ADP/ATP ratio activates AMPK, which leads to p53-mediated inhibition of p38MAPK. By activating NF-κB, p38MAPK acts as an important up-stream effector of the SASP. Therefore, in OIS a high NAD+/NADH ratio finally results in the activation of the SASP. The apparently contrasting observation in different models of senescence lends support to the hypothesis that NAD+ metabolism specifically controls distinct subsets of SASP factors. Indeed, MiDAS is triggered by a drop of NAD+/NADH and leads to IL-10 and TNF-α production, whereas OIS boosts NAD+ levels, in contrast with the general decline of NAD+/NADH ratio observed in aging and confirmed in replicative senescence.

15We can conclude that dramatic alterations of carbohydrate and lipid metabolism occur in senescent cells, with divergent outcomes partially depending on senescence trigger. In general, senescent cells acquire a “glycolytic state,” but the fate of the resulting pyruvate changes between replicative and oncogene/therapy induced senescence. Notably, the pyruvate “hub” has been promoted as a druggable target for treatment of many diseases, including diabetes, ischemic heart disease, and cancer. By directing specific substrates to one pathway or another, the entire metabolic set-up of the cell can be affected, in some cases reaching a desirable effect.

16Although they share common characteristics, ECs can exhibit several phenotypic differences, depending on the specific chemical and physical characteristics of the vascular districts in which they are living. ECs in the microvasculature are involved in the bidirectional exchange of gases, macromolecules, and cells between tissues and blood, and can also perform enzymatic modifications of circulating mediators, such as lipoproteins and angiotensin I. ECs actively participate in angiogenesis, i.e., the generation of new capillaries from existing vessels; in the adult physiological angiogenesis occurs mainly in the female reproductive system and in wound healing, however it plays a significant role in promotion and progression of many pathological conditions, including cancer and chronic inflammation. Angiogenesis implies a switch of selected ECs toward a proliferative and migratory phenotype. These ECs are known as tip and stalk cells, the first guiding the migration of the latter to achieve the elongation of the developing vessel. Angiogenesis is a high energy-demanding process, therefore, from their original quiescent state, ECs must undergo a substantial reprogramming of their metabolism.

17Quiescent ECs rely mainly on glycolysis for their energy demanding, despite the high abundance of oxygen in the vascular compartment. Accordingly, even if the mitochondrial mass in ECs is variable depending to the vascular district; it is generally lower than other cell types. During angiogenesis, vascular endothelial growth factor (VEGF) signaling induces in ECs a further boost of glycolysis, with the upregulation of the GLUT-1 transporter and an increased synthesis of the allosteric modulator fructose-2,6-bisphosphate (F2,6BP) by the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a master regulator of glycolysis. In this context, EC mitochondrial metabolism remains largely underexplored. While a substantial amount of evidence is available on the impairment of angiogenesis under glucose-deprivation conditions, only one very recent report demonstrated that also the inhibition of electron transport chain decreases EC proliferation. Interestingly, this effect is related to a lowered NAD+/NADH ratio, rather than to a decreased availability of ATP.

18The notion that ECs exhibit different glycolytic rates according to their specialization or proliferation rate is quite intuitive, however, glycolysis is not only an energy supplier for these cells but can modulate their function and phenotype. Under hypoxic conditions, the hypoxia inducible factor 1-alpha (HIF-1α)-mediated upregulation of the glycolytic pathway, coupled with the downregulation of PDH, induces an accumulation of lactate, which in turn i) stabilizes HIF-1α and mediates a paracrine proangiogenic effect on neighboring ECs, ii) affects the functional polarization of tumor-associated macrophages toward the pro-tumoral M2 phenotype. EC phenotype can be changed through targeting enzymes or regulators of the glycolysis. The forkhead box O transcription factor 1 (FOXO1) plays a major role in maintaining ECs in a quiescent state by suppressing c-myc signaling and reducing glycolysis. Similarly, the downregulation of hexokinase 2 (HK2) via the disruption of the fibroblast growth factor receptor (FGFR)/c-myc axis impairs proliferation and migration of ECs. The interplay between the two PK isoforms represents an important crossroad in determining the fate of the resulting pyruvate. In a model of pulmonary arterial hypertension, the increased PKM2 expression fosters aerobic glycolysis and induces a more proliferative state. On the contrary, under physiological conditions, the laminar blood flow promotes the NO-mediated S-nitrosylation of PKM2, which results in a reduced glycolysis and in an enhanced funneling of substrates through the PPP. In cells with a low replication rate, like quiescent ECs, a balanced PPP activity promotes antioxidant responses through synthesis of reducing equivalents in form of NADPH.

19The metabolic features of senescent ECs are still poorly explored. Moreover, whether the effects of EC senescence, including the SASP, could be mediated by metabolic changes is still being debated. An emerging role for SIRT3, a member of the sirtuin family mainly localized in the mitochondria, in cellular aging has been outlined by recent studies. Through its NAD-dependent deacetylase activity, SIRT3 regulates the mitochondrial metabolic pathways, including FAO. Specifically, SIRT3 promotes the influx of substrates into the TCA cycle by promoting the activity of PDH and acyl-CoA dehydrogenase. Moreover, SIRT3 seems to exert a positive regulator effect on the TCA cycle, even if results are still contradictory. Similarly to the other members of the sirtuin family, depletion of SIRT3 has been shown to reduce human lifespan. Notably, strategies aimed to upregulate SIRT3 in endothelial cells resulted in a higher protection against stress-induced premature senescence, an effect mediated by the deacetylation of FoxO3. For some aspects, ECs seem to escape the rules describing the senescence-associated metabolic shift that have been postulated from studies in other cell lineages. For example, the observation that HDF senescence is accompanied by an upregulation of the whole glycolytic machinery was not confirmed in HUVECs. Conversely, a recent report showed a senescence-associated decline in EC glycolysis, which is mediated by a reduced PFKFB3 activity. This trend, but not senescence, was reverted by nuclear factor erythroid 2-related factor 2 (NRF2) overexpression. Another study on EC replicative senescence revealed that a NAMPT/SIRT1/FoxO1-mediated slight increase in aerobic glycolysis exerts a protective effect by limiting ROS production.

20Glutamine represents another important metabolite for ECs. It can be used as an energy source, via deamination and subsequent transamination to form α-ketoglutarate which enters the TCA cycle, to produce the antioxidant peptide glutathione, or to provide substrates for nucleotide biosynthesis. Glutamine was shown to be required for vessel sprouting, even if the impaired angiogenesis during glutamine starvation could be rescued by asparagine supplementation. The notion that senescent ECs strongly rely on glutaminolysis for their energy demand comes from the observation in these cells of an increased lactate synthesis independent from glycolysis. Remarkably, the inhibition of glutaminase 1 (GLS1) is able to induce apoptosis and senescence even in young ECs.

21Although ECs obtain a minor amount of ATP from oxidative phosphorylation, they efficiently oxidize fatty acids: ECs are endowed with all the proteins required for the uptake and intracellular transport of fatty acids. Indeed, in the microcirculation, ECs can extract fatty acids from circulating lipoproteins through lipoprotein lipase (LPL). Fatty acids are then absorbed through the fatty acid transport proteins (FATP)3 and FATP4 and bound by the intracellular fatty acid binding protein FABP4. Fatty acids can then be oxidized to provide carbons to replenish TCA cycle, thus allowing the synthesis of dNTP precursors, or directed to the surrounding tissues. In a recent report, Kalucka et al. showed that fatty acid oxidation is the only upregulated metabolic pathway in quiescent ECs. Notably, beta-oxidation is increased neither for bioenergetic purposes nor to meet the anabolic demands of the cells. Rather, fatty acids are oxidized to increase NADPH regeneration by the malic enzyme once their carbons enter the TCA cycle in form of acetyl-CoA. As a result, quiescent ECs have higher amounts of reduced glutathione and thus are more protected against oxidative stress. Interestingly, this beneficial effect is mediated by Notch1 signaling, which exerts also a central role during the earlier phases of senescence, by switching the secretome of ECs away from the pro-inflammatory SASP and toward the TGF-β-mediated release of immunosuppressive and fibrogenic factors.

22Cellular senescence also involves endothelial progenitor cells (EPCs), a population of circulating CD34+ cells participating in new vessel formation and in vascular remodeling through their ability to differentiate into ECs. The age-related decline in EPC survival and regeneration could precipitate endothelial dysfunction, thus triggering the onset of CVDs. It has been demonstrated that the increased susceptibility of elderly individuals to ischemic disorders could be mediated, at least in part, by a blunted response of senescent EPCs to hypoxia, which fail to upregulate selected genes related to HIF-1 signaling and glucose uptake. To this regard, the upregulation of SIRT1 and NRF2 have both been shown to delay EPC senescence and to preserve the proliferative, migratory, and angiogenic activities in senescent EPCs.

23Investigations on EC metabolic features could take advantage of their peculiar localization and lend important insight into the pathogenesis of CVDs. Targeted metabolomics of large cohorts of samples are required to get a more comprehensive point of view on mechanisms which were extensively characterized in in vitro models. While multiomics approaches proved useful to identify circulating signatures with a diagnostic and/or prognostic role for many conditions, their role in providing ex vivo mechanistic clues is hampered by the lack of information on the relative contribution of various tissues to the circulating metabolome. To address this issue, the field is moving toward more specific approaches allowing the deconvolution of complex circulating signatures, including the application of machine learning tools and the study of EVs. A growing body of evidence is focusing on the characterization of endothelial EVs isolated from plasma. Even if a consensus on the methods for isolation and characterization of these EVs is far from being reached, it is important to remark that the profiling of the cargo of circulating EVs sorted according to the parent cells allows enhanced specificity compared to single molecular markers in the blood.

24Figure 2 provides an overview of the most relevant physiologic and pathologic pathways in ECs. Understanding the mutual influence between EC senescence and the complex network of metabolic pathways could provide valuable insight into the pathogenesis of many ARDs. This will be the focus of the next sections.

25Increasing evidence is suggesting a bidirectional interplay among EC metabolism and ARDs. One of the most characterized metabolic alteration in ECs exposed to hyperglycemia, i.e., a known senescence-promoting stimulus, is the diversion of glycolytic intermediates into alternate pathways (Figure 2). This is the result of the activation of the enzyme polyADP-ribose polymerase 1 (PARP1) following the ROS-induced DNA damage. PARP1 mediates the ribosylation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and depletes NAD+ intracellular levels, further inhibiting the glycolysis flux. Moreover, entry of glucose 6-phosphate into the PPP is also restricted, due to a cAMP-mediated impairment of glucose 6-phosphate dehydrogenase during hyperglycemia. The reduced PPP activity leads to lowered NADPH levels, causing both a reduced endothelial nitric oxide synthase (eNOS) activity and a blunted antioxidant response. In addition, excessive glucose is metabolized into the pathologic polyol and hexosamine biosynthetic pathways, which are the major contributors to the generation of advanced glycation end-products in diabetes. The aberrant non-enzymatic glycation of circulating and intracellular proteins is a key determinant of T2DM cardiovascular complications. Endothelial dysfunction (ED) and senescence are “historically” considered two consequences of T2DM, possibly as a result of the overabundance of senescence-promoting, endothelium-damaging factors in the bloodstream of these patients, e.g., pro-oxidant molecules, glucose, and lipids. In particular, the role of EC senescence has been emphasized in both the development of T2DM and of its CV complications. Indeed, EC senescence has been found in the adipose tissue of obese subjects preceding the development of T2DM, as well as in other vascular districts in diabetic mice and humans. On the other side, the observation that ED development often precedes the appearance of the T2DM phenotype in obese subjects, coupled by the intuitive assumption that the endothelium represents the biggest organ regulating nutrient availability across the whole organism, has prompted the research toward the study of the effect of selective gain/loss of function of metabolic genes in ECs.

26A seminal paper suggested that deletion of insulin receptor (IR) in ECs is sufficient to alter the expression of eNOS and endothelin-1 in mice treated with a high-salt diet, providing the first link among EC metabolism and general vascular tone. Later, another group showed that EC-specific Irs2 KO mice have a blunted insulin-dependent glucose uptake in the muscles when fed with HFD. Beyond insulin signaling, also the selective modulation in ECs of genes involved in angiogenesis, i.e., VEGF/VEGFR2, ANG2/TIE2, and DLL4/NOTCH1, as well as in fatty acids transport, i.e., VEGF-B/NRP1 and CD36, affect whole body metabolism. Interestingly, both promotion and inhibition of angiogenesis have been shown to foster insulin sensitivity, suggesting a context-dependent effect. Proposed mechanistic explanations include the induction of apoptosis in dysfunctional adipocyte (for restricted angiogenesis) and “healthy” expansion of adipose tissue (for enhanced angiogenesis).

27While the role of senescent EC metabolism has not been studied in relation to specific ARDs, a key role for an alteration of metabolism in senescent ECs may be inferred by studies modulating key factors involved in the senescence process. Indeed, mice with EC-specific p53 deficiency fed a high fat diet (HFD) showed improvement of insulin sensitivity and less fat accumulation compared to WT mice. Of note, p53 is a master transcription factor promoting senescence in virtually all cell types. Similarly, NF-κB activation is held to be a cornerstone of SASP development in a plethora of different SCs, including ECs. Transgenic mice expressing dominant-negative IκB under the Tie2 promoter/enhancer (thus with functional inhibition of NF-κB signaling specifically in ECs) were protected from the development of insulin resistance associated with both genetic and diet-induced obesity. Strikingly, these mice showed also an increase in lifespan, coupled by a decreased age-related insulin resistance and vascular senescence. The relevance of these findings for human ARDs remains to be tested. However, an increased expression of p53, along with an increased activation of NF-κB has been observed in aged arteries from human subjects.

28Beyond T2DM and vascular function, also heart remodeling has been shown to be affected by metabolic alterations in ECs. Indeed, genetic ablation of Rbp-jκ (Notch signaling) in ECs promoted alterations in fatty acid metabolism in the whole organism, followed by heart hypertrophy and failure. The deprivation of available FFAs as substrate prompted the use of glucose in cardiomyocytes and the consequent activation of the mTOR pathway, a master metabolic rheostat regulating both cellular senescence and organismal aging. Accordingly, a ketogenic diet was able to restore normal cardiac function in this mouse model, suggesting the potential of dietary intervention to treat (and not only prevent) also life-threating ARDs. A similar framework has been proposed also to explain the cardioprotective properties of sodium-glucose cotransporter (SGLT)-2 inhibitors (i). SGLT-2i are a recently introduced class of glucose-lowering drugs inhibiting the reabsorption of glucose in the proximal convoluted tubule, thus promoting glucose elimination through the kidneys. Clinical trials have shown a striking benefit in terms of CV mortality and worsening of heart failure in diabetic patients treated with SGLT-2i, an effect not ascribable to an improved glycemic control. It has been hypothesized that the decreased availability of glucose, coupled by an increase in the circulating levels of ketone bodies (KB), improves the energetic function of the heart. This hypothesis has been recently tested in porcine, non-diabetic hearts, where the treatment with a SGLT-2i switches myocardial fuel utilization away from glucose toward KB, FFA, and branched chain amino acids, thereby improving myocardial energetics, enhancing left ventricular systolic function, and ameliorating adverse left ventricle remodeling.

29The promotion of myocardial function by shifting toward FFA utilization is likely dependent on the function of the liver, rather than EC metabolism. However, EC metabolism may influence the pathobiology of different organs by at least 3 mechanisms: (i) by releasing active secondary mediators, such as NO; (ii) by regulating the flow of lipids from bloodstream to organs; (iii) by regulating vessel density, and thus indirectly nutrients availability and interstitial insulin levels. Beyond adipose tissue and muscle insulin sensitivity, the latter mechanism has been shown to be crucial for the development of age-induced osteoporosis. Indeed, skeletal blood flow and endothelial Notch activity are reduced in aged mice, leading to decreased angiogenesis and osteogenesis, which can be reverted by genetic reactivation of Notch. As mentioned above, Notch is regarded as a central metabolic sensor and regulator in multiple cell types.

30Regarding possible secondary mediators released by ECs, the role of EVs deserves particular attention. The effects of EVs are now attracting intense interest also in the context of aging and ARDs. For instance, senescent ECs secrete miR-31 enriched EVs to inhibit mesenchymal stem cells osteogenic differentiation, possibly contributing to age-induced osteoporosis. Of note, miR-31 is upregulated by high-glucose and inhibits cell differentiation also in other contexts.

31Overall, increasing evidence is suggesting a key role for EC metabolism in a plethora of ARDs, while less studies are available regarding the effect of the specific alterations of senescent EC metabolism in the whole organisms. However, the emerging roles of endothelial p53 and NF-κB, two cornerstones of the senescence process, in the regulation of both cellular and organismal metabolism suggest that metabolic alterations in specific senescent cells, and in particular ECs, can affect whole body metabolism and ARD development, a hypothesis deserving exploration in the future.

32Thirty years ago, the identification and cloning of VEGF paved the way to the development of novel strategies aimed to treat those conditions in which angiogenesis plays a dominant role. Agents targeting members of the VEGF family and their receptors are currently routinely employed in the treatment of many solid malignancies, including colorectal cancer, renal cell carcinoma, and non-small cell lung carcinoma, and in non-neoplastic conditions with a recognized angiogenic component, such as proliferative diabetic retinopathy and age-related macular degeneration. Investigations on the mechanism of action of anti-VEGF agents helped to highlight the aforementioned interesting connections between angiogenesis and EC metabolism and offered a number of novel druggable targets that could prove useful, for example, in the case of failure of anti-VEGF treatments.

33Targeting the high-glycolytic state of ECs within tumor vessels is emerging as an anti-cancer therapeutic strategy. In a murine model, pharmacological inhibition of glycolysis activator PFKFB3 promotes the normalization of tumor vessels and facilitates delivery of chemotherapeutic drugs. Moreover, PFKFB3 tightened ECs by upregulating vascular endothelium (VE)-cadherin, thus reducing the passage of cancer cells across the EC monolayer. Interestingly, inhibition of PFKFB3 lowered the expression of adhesion molecules in ECs treated with IL-1β, suggesting that limiting glycolysis could represent a feasible approach to prevent the SASP-mediated spreading of EC senescence. PFKFB3 inhibitors recently reached the clinical trial phase, with promising results from a phase 1 multi-center study conducted on patients with solid tumors.

34Given the proangiogenic role of lactate in ECs, several studies focused on target the monocarboxylate transporter 1 (MCT1) to avoid lactate exchange across ECs. Lactate mediates angiogenesis through the activation of the NF-κB/IL-8 pathway and the stabilization of HIF-1α. Evidence on cell and animal models revealed that MCT1 inhibition in ECs can drive direct anti-angiogenic effects through the enhanced degradation of HIF-1α. Drugs targeting MCT1 in a non-selective manner are currently been tested in small-scale trials. On the other hand, administration of the telomerase activator TA-65 contributes to improve blood flow recovery through increasing expression of HIF-1α, VEGF-A, and peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1-alpha (PGC-1α), indicating that telomerase activation could prove a valuable therapeutic option to rescue ischemic tissues in elderly individuals.

35Targeting EC metabolism could prove useful in treating a plethora of conditions sharing endothelial dysfunction as a pathogenic mechanism. Evidence of an age-related impairment of endothelial function dates back to 1990s. The identification of eNOS as the enzyme responsible for NO synthesis prompted the supplementation of its precursor L-arginine to reverse endothelial dysfunction. However, the enthusiasm for this essential amino acid as an easy therapeutic option in the prevention of many CVDs was tempered by the observation that L-arginine could even exert detrimental effects on vascular function through an inductive effect on arginase, which competes with NOSs for their common substrate. For this reason, attention has moved to arginase as a putative target to ameliorate age-related endothelial dysfunction. A major contribution of its activity in determining microvascular dysfunction and remodeling has been outlined in obesity, arterial hypertension, and T2DM. Interestingly, knockout of the gene encoding for the mitochondrial arginase type II (Arg-II) has been shown to restore eNOS function, to counteract the SASP in senescent ECs, and to extend lifespan in mice through the inhibition of mTOR signaling. Moreover, mTOR blockade with rapamycin decreases the expression of the arterial senescence marker p19 and ameliorates oxidative stress- mediated endothelial dysfunction in old mice, suggesting a possible role for the rapamycin analogs, i.e., rapalogs, in the treatment of age-related CVDs.

36Fenofibrate, a PPAR-α agonist, is a common lipid-lowering drug exerting a number of interesting pleiotropic effects. By increasing EC eNOS expression and lowering circulating oxidized LDL, fenofibrate ameliorated the age-related endothelial dysfunction in a cohort of healthy individuals. Further mechanistic studies on animal models revealed that the eNOS stimulation, along with other fenofibrate effects, is mediated by enhanced AMPK activity following liver kinase B1 (LKB1) translocation from the nucleus to the cytoplasm. Additionally, fenofibrate lowered the cyclooxygenase 2-mediated production of vasoconstrictor prostaglandins. By restoring the balance between vascular relaxation and contractility, fenofibrate could represent a feasible preventive approach for the vascular complications of T2DM. Notably, fenofibrate ameliorated osteoarthritis in elderly patients by selectively clearing senescent chondrocytes. The observation of a similar senolytic effect on ECs would provide additional benefits for this largely employed drug also in the treatment of a number of age-related vascular conditions.

37Investigations into a variety of diseases highlighted a role for CPT1, the rate-limiting enzyme for FAO, as a druggable target. Inhibition of endothelial CPT1a impairs EC proliferation and activates the endothelial-to-mesenchymal transition, which plays a role in the pathogenesis of pulmonary arterial hypertension, atherosclerosis, and tumor spreading. On the contrary, stimulation of CPT1 activity by chronic L-carnitine administration improved endothelial function in an animal model of congenital heart defect. In light of the crucial role of FAO in maintaining EC quiescence and redox balance, L-carnitine supplementation, by counteracting the age-related decline in CPT1a activity, could prove useful to delay the onset of age-related endothelial dysfunction. The encouraging evidence from several in vitro and animal studies still needs to be supported by large clinical trials.

38Glutamine, the most abundant circulating amino acid, has been extensively studied in aging research. Its blood levels decline during acute illness are increased in healthy centenarians. A seminal study showed that glutamine administration in rats improves eNOS activity and reduces the endothelial inflammatory response following cardiopulmonary bypass. The activity of glutamine synthetase is impaired during endothelial dysfunction, due to peroxynitrite-mediated nitration of its active site. This could lead to reduced TCA cycle anaplerosis, which results in the impairment of EC antioxidant system (see above). Of note, restoring the glutamine-dependent anaplerosis through GLS overexpression has already proved beneficial in ameliorating the age-related bone loss, and in delaying EC senescence. Finally, the evidence that glutamine metabolism is also involved in EC proliferation supports the hypotheses that targeting GLS1 could represent a feasible approach to treat diseases associated with an aberrant EC proliferation, and that glutamine supplementation can foster endothelial progenitor cell mobilization and promote vascular endothelium repair in diabetes-related ischemic injury.

39The observations discussed in the present section are summarized in Table 1. Altogether, these evidences reinforce the notion that alterations in EC metabolism are rather primary drivers than consequences of disease. The development of drugs capable of targeting specific enzymes or pathways at the endothelial level is still in its infant phase. However, their progression into large clinical trials is imminent and could represent a turning point in the treatment of CVDs.

40Accumulating evidence is suggesting that SCs are characterized by a deep reshaping of metabolic pathways. The overall picture appears highly complex, considering that specific metabolic alterations characterize different cell types and various pro-senescence stimuli. However, a general trend toward a more glycolytic state in senescent cells has been reproduced with different stimuli and in a wide range of cell types, including ECs. The role of EC metabolism in the development of T2DM and CVDs is clearly emerging, mainly thank to specific mouse models with tissue-selective deletion of metabolic genes. However, less information is available regarding the role of senescent EC metabolism in ARDs development, even though preliminary findings suggest a key role for senescence EC related genes, e.g., p53 and NF-κB, in the regulation of organismal aging. Disentangling the effective contribution of altered EC metabolism in humans remains challenging, as well as the impact of dietary or pharmacological intervention on this very specific process. Studies focusing on senescent EC metabolism are warranted to clarify the disease-modifying/preventive potential of both these approaches. In fact, interventions aimed at modifying diet and metabolism have already proven to be potentially effective strategies in the prevention and treatment of ARDs. In particular, the preventive and curative roles of specific diets have now been demonstrated by placebo-controlled, randomized clinical trials showing a cardioprotective role for Mediterranean diet and the reduction of multiple cardiometabolic risk factors with CR. While future studies will define to what extent these effects can be obtained with specific molecules/nutrients, the quality of evidence regarding these dietary patterns and ARD prevention should prompt the adoption of a Mediterranean-based, low-calories dietetic regimen at the population level.

41JS, FP, and AG collected relevant literature and wrote the manuscript. JS and AG prepared the figures. FO, AP, and MR made a substantial, direct, and intellectual contribution through their experience in the field. MR critically advised and reviewed the manuscript.

42The 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.