Impacts of aging and fluid shear stress on vascular endothelial metabolism and atherosclerosis development

Senescent ECs exhibit upregulation of glycolysis [19, 32], which may initially support energy requirements of migration and proliferation [19], but becomes maladaptive under pathological conditions. Hyperactivation of glycolysis contributes to dysfunctional EC proliferation, aberrant migration, and pathological angiogenesis, all of which promote plaque formation and instability [33]. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a crucial glycolytic regulator, converts fructose-6-phosphate to fructose-2,6-bisphosphate (F2,6BP), enhancing PFK-1 activity and thus promoting EC proliferation, migration, and angiogenesis [23]. Moreover, elevated PFKFB3 expression is commonly observed in vulnerable plaque regions [34]. Studies showed that inhibition of PFKFB3 partially reduces glycolysis, and its inhibition was shown to normalize EC behavior, mitigate pathological angiogenesis, and promote atherosclerotic plaque stability [24, 34–36]. Moreover, PFKFB3-mediated glycolysis promotes EC inflammation, particularly in response to atherogenic stimuli such as lipoprotein(a) (Lp(a)) and tumor necrosis factor (TNF)-α [37, 38], and contributes to EndMT, a key event in atherogenesis [39]. Hypo-glycolysis metabolism in ECs may also contribute to endothelial dysfunction in atherosclerosis. Xu et al. found that microRNA (miR)-143, which inhibits key glycolytic enzymes like HK2, LDHA, and PKM2, was upregulated in atherosclerotic plaque. Overexpression of miR-143 significantly decreases glucose consumption and ATP formation. This energy deficit directly impairs crucial EC functions. [40]. Similarly, Yang et al. demonstrated that disturbed flow activates the protein kinase, AMP-activated, alpha 1 (PRKAA1)/AMP-activated protein kinase α1 (AMPKα1)-hypoxia-inducible factor α (HIFα)-glycolysis axis that supports EC proliferation, and inhibition of PRKAA1 accelerates atherosclerosis in ApoE^−/− mice [41]. These results suggest that both hyper- and hypo-glycolysis disrupt EC homeostasis, and a glycolytic balance is essential for vascular health and atherogenesis. However, the detailed mechanisms by which glycolysis and glycolytic intermediates mediate atherosclerosis progression remain incompletely understood.

Lactate, derived from glycolysis and glutamine catabolism, functions as a crucial carbon source for cellular metabolism and a key signaling molecule [42]. Elevated lactate levels are associated with various inflammatory diseases and serve as prognostic indicators in CVDs [43–45]. In atherogenesis, enhanced glycolysis further increases lactate production and release [46], which leads to extracellular acidification, increases EC permeability, and accelerates atherosclerosis progression [47]. In addition to these indirect effects, lactate also directly modulates vascular inflammation and angiogenesis by activating nuclear factor (NF)-κB and HIF-1α pathways [48–52]. Xu et al. demonstrated that lactate promotes PFKFB3-driven angiogenesis through AKT phosphorylation [22], while lactate also induces EC permeability via extracellular signal-regulated kinase (ERK)-dependent activation of calpain1/2 to disrupt vascular endothelial (VE)-cadherin [53]. Moreover, lactate contributes to epigenetic reprogramming via histone lactylation. Zhang et al. demonstrated that glycolysis-derived lactate can modify histone by adding lactyl groups to become histone lysine (K) residues, a process known as "lactylation," which influences gene transcription and epigenetic modifications [54]. Dong et al. reported that lactate-dependent H3K18 lactylation (H3K18la) promotes the EndMT and atherosclerosis progression [55]. In addition to histones, lactate also induces lactylation of snail, a transforming growth factor (TGF)-β transcription factor that activates the TGF-β/Smad2 signaling pathway, further promoting the EndMT [56].

Interestingly, lactate may also have protective roles. Exercise is recognized as an atheroprotective factor and is known to generate significant amounts of lactate. Exercise-induced lactate activates G protein-coupled receptor 81 (GPR81), also known as hydroxy carboxylic acid receptor 1 and a selective lactate-sensing receptor, which is downregulated under atherogenic OS. Activation of GPR81 prevents OS-induced inflammation through the ERK5/Krüppel-like factor 2 (KLF2) signaling pathway [57]. In addition, lactate-induced lactylation of methylated CpG-binding protein 2 represses epiregulin/mitogen-activated protein kinase (MAPK) signaling, inhibiting EC inflammation and atherosclerosis progression [58]. Thus, lactate plays a dual role in atherosclerosis, being both a detrimental and potentially beneficial factor.

Glycolysis produces pyruvate, which typically enters mitochondria and is metabolized by pyruvate dehydrogenase (PDH) to acetyl-CoA, subsequently entering the tricarboxylic acid (TCA) cycle in physiological conditions. However, during hyperactive glucose metabolism, pyruvate can be atypically metabolized into free acetate instead of acetyl-CoA [59]. Zhu et al. demonstrated that this acetate accumulation promotes the EndMT, contributing to atherosclerosis [60]. Paradoxically, some research suggested that increasing acetyl-CoA levels via acetate supplementation might suppress TGF-β-induced EndoMT. This indicates a complex and context-dependent role for acetate [61]. Collectively, acetate may also exert beneficial effects in the aging endothelium by rescuing epigenetic homeostasis. These results indicate that acetate counteracts aging-associated endothelial dysfunction by epigenetic rescue.

Taken together, these findings highlight that dysregulated glycolytic intermediates, particularly acetate and lactate, play dual and context-dependent roles in modulating endothelial aging and atherosclerosis.

In ECs, a portion of glucose enters alternative metabolic routes beyond classical glycolysis. These side pathways—namely the PPP, HBP, and polyol pathway—play critical roles in regulating redox balance, protein glycosylation, and metabolic stress responses.Pentose phosphate pathway. The PPP is the primary cellular source of NADPH, which supports reductive biosynthesis, antioxidant defense, and endothelial nitric oxide synthase (eNOS)-mediated nitric oxide (NO) production [62]. NADPH is also essential for glutathione reductase to regenerate reduced GSH, maintaining intracellular redox homeostasis [19, 63]. Glucose-6-phosphate dehydrogenase (G6PD), the enzyme of the PPP, was shown to enhance NADPH and NO production while reducing ROS in ECs under oxidative stress [64]. Conversely, G6PD inhibition lowers NADPH and NO levels, leading to EC dysfunction [39, 65, 66]. A G6PD deficiency is associated with increased cardiovascular risks [67, 68], particularly in elderly populations [69]. Nevertheless, the precise mechanisms through which the PPP and G6PD regulate EC dysfunction and atherogenesis during aging remain unclear and warrant further investigation.Hexosamine biosynthesis pathway. Within the HBP, fructose-6-phosphate is converted into glucosamine-6-phosphate and is subsequently metabolized into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a crucial metabolite for O- and N-linked protein glycosylation, supporting various EC functions and cardiovascular pathology [70, 71]. HBP activity exerts context-dependent effects: acute activation may offer cardioprotection, whereas chronic HBP upregulation under hyperglycemia induces endoplasmic reticular (ER) stress, lipid accumulation, and inflammation [72]. Overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP, was implicated in metabolic stress and atherosclerosis under a hyperglycemic condition [73]. Enhancing the HBP leads to increased UDP-GlcNAc and O-linked glycosylation, which impair eNOS activity [74]. Although emerging evidence suggests that both aging and chronic hyperglycemia activate the HBP in ECs, the direct impact of age-associated HBP remodeling on vascular dysfunction remains insufficiently characterized.Polyol pathway. Under physiological conditions, only approximately 3% of glucose enters the polyol pathway. However, in aging and hyperglycemic states, its activity is markedly enhanced. This pathway, catalyzed by aldose reductase (ALR2) and sorbitol dehydrogenase (SORD), converts glucose to fructose, consuming NADPH–a crucial antioxidant. Excess fructose may generate 3-deoxyglucosone (3-DG), a precursor for harmful advanced glycation end products (AGEs) [19, 75, 76]. AGEs not only suppress eNOS expression but also trigger oxidative stress via the MAPK/ERK pathways [77, 78] While largely inactive under normal conditions, hyperglycemia activates the polyol pathway in ECs, leading to detrimental consequences [79].

In summary, both aging and atherosclerosis promote increased glycolysis and the accumulation of intermediates and metabolites from glycolysis and its side pathways, leading to EC dysfunction. Elucidating the age-specific regulation of these pathways and their metabolite-mediated signaling mechanisms may provide novel targets for preventing or reversing endothelial dysfunction in atherosclerosis.

In ECs, long-chain FAs (LCFAs, consisting of 12–20 carbons) are taken up from the circulation and transported across plasma membranes by a set of specific proteins, including lipoprotein lipase (LPL), glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), cluster of differentiation 36 (CD36), FA transport proteins (FATPs), and FA-binding proteins (FABPs) [81]. Among these, CD36 is the most well-characterized FA transporter. EC-specific CD36-knockout enhances glucose utilization, whereas a CD36/low-density lipoprotein receptor (LDLR) deficiency reduced atherosclerotic lesion formation in a murine model [82]. Similarly, FABPs, particularly FABP4, play essential roles in mediating endothelial-specific LCFA trafficking to specific intracellular sites for oxidation or storage in lipid homeostasis [83]. Key signaling pathways, including peroxisome proliferator-activated receptor γ (PPARγ) and Notch, regulate FABP4 expression to facilitate transendothelial FA transport [84, 85]. Conversely, downregulation of FABP4 by apelin/apelin receptor (APLNR)-mediated Forkhead box O1 (FOXO1) suppression in ECs limits excessive FA transport and tissue accumulation, contributing to atheroprotecton against vascular diseases such as atherosclerosis [86–88]. Pharmacological inhibition of FABP4 with compounds such as BMS309403 enhances eNOS expression and improves EC function, reducing plasma triglyceride (TG) levels and atherosclerosis progression in diabetic models [89, 90]. In the context of aging, senescent ECs show increased expression of FA transporters, such as CD36 and FABP, suggesting a metabolic shift from glycolysis to FA oxidation to support a proinflammatory phenotype [91]. These findings highlight how age-related shifts in FA transporter expression contribute to EC dysfunction.

Once inside a cell, FAs are esterified into TGs and stored within LDs, dynamic organelles derived from the ER. The biosynthesis and turnover of LDs are regulated by enzymes such as diacylglycerol O-acyltransferase (DGAT) and adipose TG lipase (ATGL) [92]. Moreover, autophagy components are involved in LD biogenesis. During prolonged starvation, autophagy breaks down membrane organelles to release FAs, which are re-esterified and sequestered into newly formed LDs for energy storage. Conversely, when cellular energy demands increase, LDs can be selectively degraded through autophagy-dependent lipophagy to release FAs for beta-oxidation and ATP production [93, 94].

In vascular ECs, LD accumulation is influenced by FA availability and DGAT1 and ATGL activities. For example, treatment with oleic acid promotes LD formation and triggers compensatory lipolysis through ATGL. Inhibition of ATGL delays FA release from LDs. LD-derived FAs reduce saturated FA-induced ER stress and enhance FAO, thereby reducing glycolysis to meet metabolic demands under normal conditions [95]. Although transient LD accumulation may serve as a protective buffer, chronic LD accumulation in ECs leads to decreased NO production, increased NF-κB signaling, elevated blood pressure, and accelerated atherosclerosis, particularly in ATGL-deficient or high-fat diet-fed mice [96, 97]. Inhibition of LD formation using a DGAT inhibitor restores NO production in ATGL-deficient mice [98]. Moreover, loss of ATGL1 triggers TG accumulation and ER stress-induced inflammation in ECs, contributing to plaque formation [99]. A high-fat diet induces LD accumulation, which impairs EC ciliation through stearoyl-CoA desaturase 1 (SCD1), promoting atherosclerosis [100].

While LDs are increasingly recognized as aging-associated features, their precise role in human vascular aging is not fully defined. Aged macrophages with dysfunctional autophagy exhibit increased LDs and lose their ability to regress atherosclerosis [101, 102]. In senescent ECs, increased LD accumulation was linked to elevated expressions of proinflammatory cytokines [91]. These findings highlight the impacts of neutral lipid and LD accumulation in ECs, suggesting that disrupted LD dynamics in ECs may contribute to vascular pathologies.

FAO is a mitochondrial process where FAs are converted into acetyl-CoA to provide energy for the TCA cycle and produce ATP. Although ECs primarily rely on glycolysis for ATP production, quiescent ECs exhibit increased FAO to support mitochondrial respiration and maintain a redox balance by producing NADPH [103]. In addition, senescent ECs display heterogenous metabolic reprogramming. While some studies reported increased glycolytic activity, lactate production, and enhanced TCA cycle activity and mitochondrial respiration during senescence [32], others showed a marked shift toward FAO and a reduced glycolytic rate and lactate production in senescent ECs compared to young ECs [91]. This metabolic shift may vary by cell type and mode of senescence induction [104]. Moreover, this metabolic shift from glycolysis to FAO in senescent ECs is closely associated with increased secretion of proinflammatory factors characteristic of the senescence-associated secretory phenotype (SASP), which exacerbates chronic vascular inflammation (often referred to as inflammaging) and contributes to vascular diseases such as atherosclerosis and hypertension [105]. Pharmacologic inhibition of carnitine palmitoyl transferase (CPT)1, the rate-limiting enzyme in FAO, using etomoxir attenuated SASP-related cytokine expression, suggesting a causal relationship between excessive FAO and EC inflammation during aging [3, 91]. FAO also plays a role in deoxynucleotide triphosphate (dNTP) synthesis and EC proliferation; silencing CPT1a decreases EC proliferation and metabolic homeostasis [106]. The role of increased FAO during senescence in promoting pathological angiogenesis remains unclear and warrants further investigation. Given the complexity of FAO regulation, which is influenced by the FA type, cellular environment, and coexisting risk factors, its contribution to atherosclerosis and therapeutic potential should be explored in greater depth.

De novo FA synthesis (de novo lipogenesis (DNL)) is vital for EC homeostasis, by regulating membrane formation, lipid signaling, and metabolic balance. ECs express key enzymes for DNL, including ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and FA synthase (FASN), which convert citrate into long-chain FAs (LCFAs) such as palmitate [107]. With aging and metabolic disorders such as diabetes, DNL is disrupted. FASN expression decreases in EC-enriched tissues of diabetic mice, whereas insulin restores its levels and eNOS activity, highlighting the importance of DNL in glucose-sensitive endothelial regulation. In addition, FASN inactivation results in impaired eNOS palmitoylation, increased vascular permeability and inflammation, and defective angiogenesis, further reinforcing its regulatory role in EC health [108]. Aging and metabolic stress disrupt DNL in ECs, with senescent cells exhibiting decreased FASN expression, elevated inflammation, and loss of endothelial function [91]. Conversely, higher FASN expression may lead to pathological angiogenesis through malonyl-CoA-mediated inhibition of mammalian target of rapamycin (mTOR) complex 1 (mTORC) signaling [109]. This paradox underscores the complexity of FASN’s role in EC biology, suggesting that both excessive and insufficient DNL can contribute to endothelial dysfunction, depending on the context and temporal dynamics.

Sphingolipid metabolism is a closely related lipid pathway that produces ceramide and sphingosine-1-phosphate (S1P), which have contrasting effects (beneficial vs. detrimental) in many cellular processes. Aging skews the ceramide/S1P ratio in favor of ceramide accumulation, leading to increased oxidative stress, apoptosis, and impaired NO bioavailability [110]. The imbalance, known as the sphingolipid rheostat, contributes to atherosclerosis progression and plaque instability [111]. Interestingly, in a mouse model of coronary atherosclerosis, hemodynamic stress was shown to induce changes in sphingolipid metabolism that favored the production of S1P over ceramides, suggesting that blood vessels may be adaptative to stress [112].

In summary, aging-induced alterations in FA metabolism may disrupt EC function by reducing NO bioavailability, increasing oxidative stress, and promoting chronic inflammation. These metabolic disturbances contribute to the development of atherosclerosis. The precise mechanisms linking age-related FA metabolic reprogramming to EC dysfunction remain to be elucidated by further investigations. Understanding and targeting FA metabolic pathways in ECs may provide new therapeutic strategies for CVDs.

l-Arg is a conditionally essential amino acid involved in NO production, the urea cycle, and synthesis of multiple biomolecules [115, 116]. In ECs, l-Arg is converted to NO and L-citrulline by eNOS, a process crucial for vascular health [117]. However, in senescent ECs, eNOS activity declines, leading to impaired NO production and dysfunction [9, 118, 119]. The recycling of citrulline back to l-Arg via ASS/ASL (the citrulline-NO cycle) pathway sustains NO production even under low plasma l-Arg availability [120]. In addition to NOS, l-Arg can be catalyzed by arginase to produce urea and l-ornithine [117]. In aged or atherosclerosis-prone vasculature, arginase is upregulated, competing with eNOS for l-Arg, further reducing NO production and promoting EC dysfunction and atherosclerosis [121, 122]. Thus, arginase is emerging as a therapeutic target in vascular aging and atherosclerosis [123, 124].

In addition to NO synthesis, l-Arg can also be catalyzed by arginine decarboxylase to produce agmatine [117], which improves EC function by enhancing eNOS activity, reducing mitochondrial ROS levels, and alleviating lipid accumulation through the AMPK/phosphatidylinositol 3-kinase (PI3K)/Akt/eNOS signaling pathway [125, 126]. Meanwhile, posttranscriptional methylation of l-Arg residues in proteins generates asymmetric dimethylarginine (ADMA), a metabolic l-Arg analog byproduct and a natural NOS inhibitor that competes with l-Arg for binding, leading to NOS uncoupling (Fig. 4), EC dysfunction, and ultimately CVDs [127]. Elevated ADMA levels inhibit NO production, promote NOS coupling, and drive oxidative stress and vascular inflammation. Oxidative stress further enhances ADMA accumulation by suppressing dimethylarginine dimethylaminohydrolase (DDAH, ADMA hydrolase) activity, exacerbating EC dysfunction [128, 129]. Moreover, elevated plasma ADMA levels are a recognized hallmark of atherosclerosis [130]. NOS uncoupling is a pathological state in which NOS shifts from producing NO to generating superoxide (O₂⁻), an ROS. This shift contributes to oxidative stress, EC dysfunction, and atherosclerosis progression, particularly under cardiovascular risk factors such as diabetes, hypertension, dyslipidemia, smoking, and aging [131, 132]. ADMA-induced NOS uncoupling is closely linked to one-carbon metabolism, where S-adenosylmethionine (SAM) donates a methyl group for ADMA synthesis and is converted into S-asenosylhomocysteine (SAH), an inhibitor of methyltransferase [133]. SAH is then hydrolyzed to homocysteine, which suppresses DDAH activity, leading to ADMA accumulation [134]. A decreased SAM/SAH ratio, reflecting impairment, is associated with an increased risk of atherosclerosis in the elderly and may serve as a diagnostic biomarker [135, 136].

Moreover, tetrahydrobiopterin (BH₄), a crucial eNOS cofactor, is oxidized to dihydrobiopterin (BH₂) under oxidative stress, leading to eNOS coupling [131]. The cellular ratio of BH₄/BH₂ affects eNOS uncoupling and is implicated in the pathophysiology of EC dysfunction [137]. In senescent ECs, an elevated BH₂/BH₄ ratio exacerbates oxidative stress and inflammation [138]. Strategies targeting BH₄ regeneration via guanosine triphosphate cyclohydrolase (GPTCH) or the BH₂-reducing enzyme, dihydrofolate reductase (DHFR), may help preserve eNOS function [139, 140]. Notably, a BH₄ deficiency lowers the glutathione (GSH)/glutathione disulfide (GSSG) redox ratio, promotes eNOS S-glutathionylation, and further impairs NO production, thereby contributing to endothelial dysfunction and vascular inflammation with aging [141, 142]. Disruptions of both BH₄ availability and the GSH/GSSG balance are key mechanisms driving eNOS uncoupling during vascular aging.

Collectively, dysregulated l-Arg metabolism is the underlying cause of NO deficiencies, oxidative stress, and vascular aging. Although l-Arg supplementation has shown mixed results in clinical studies [113], maintaining a balanced l-Arg/NO pathway remain a promising therapeutic focus.

Glutamine is the most abundant non-essential aa in circulation, providing up to 70% of the TCA cycle’s carbon for ECs, a contribution comparable to that of glucose [143]. ECs metabolize glutamine via glutaminase 1 (GLS1) and glutamate dehydrogenase (GLUD) to α-ketoglutarate, which supports anabolic processes including nucleotide, protein, polyamine, glucosamine, and de novo serine synthesis [144]. Disruptions in glutamine-glutamate homeostasis are associated with CVDs [145]. Moreover, glutamine metabolism impacts other critical EC functions by inhibiting l-Arg regeneration from L-citrulline [73, 146, 147]. Notably, increased glutamine metabolism also supports the inflammatory state of macrophages within atherosclerotic lesions [148]. These findings suggest that age-related alterations in EC glutamine metabolism could contribute to EC dysfunction and atherosclerosis progression by affecting EC proliferation, the redox balance, and inflammatory responses. However, the specific mechanisms underlying these responses remain to be investigated.

Pyroglutamic acid (PCA), also known as 5-oxoproline, is a cyclic derivative of glutamic acid (or glutamine) and a gamma-glutamyl cycle metabolite indicative of glutathione turnover [149]. While glutamine’s role in atherosclerosis is being actively investigated, direct research specifically focusing on PCA’s independent role in the pathogenesis of atherosclerosis is limited. In this context, its importance seems to be more related to its involvement in the glutathione synthesis pathway and as a marker of altered glutamine/glutamate metabolism.

The three BCAAs of valine, leucine, and isoleucine are catabolized by BCAA aminotransferase (BCAT) and branched-chain α-ketoacid dehydrogenase (BCKD) enzymes into intermediates like acetyl-CoA and methylmalonay-CoA, which enter the TCA cycle [113, 150]. Age-related metabolic disorders such as hyperglycemia and hyperlipidemia elevate plasma BCAA levels, thereby accelerating atherosclerosis [151]. In senescent cells, this BCAA accumulation may be due to upregulated BCAA transporters (SLC6A14/15) or reduced BCAT/BCKD activity, which compromises antioxidant defenses by impairing glutathione synthesis and enhances oxidative stress and inflammation via mTORC and NF-κB signaling pathways [152–155]. While elevated systemic BCAA levels were correlated with increased atherosclerosis risks, the direct role of BCAA metabolism within ECs during atherogenesis, particularly with aging, remains unclear. There is evidence that BCAAs can promote EC dysfunction by increasing oxidative stress and inflammation, but the specific enzymes, transporters, and pathways involved as well as the context-dependent effects require further investigation to fully elucidate their contribution to atherosclerosis development.