Julie K. Freed

Acute consumption of a long-chain fatty acid-rich meal increased circulating ceramides in a sex-specific manner, with elevations observed only in females. Despite this, in vivo microvascular endothelial function was similarly impaired after both medium- and long-chain fatty acid-rich meals, with no differences observed between sexes. Further studies are needed to define the role of ceramide-induced microvascular dysfunction and the long-term cardiovascular impact of repeated exposure.

This study demonstrates that the metabolic phenotypes induced by 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure in rodents, used as an experimental model of menopause, strongly depend upon diet formulation. VCD-treated mice fed a corn/soy-based diet (PicoLab 5L0D) exhibited suppressed resting and total energy expenditures and increased adiposity, which parallels effects of menopause documented in humans. In contrast, VCD failed to induce such phenotypes in mice fed a wheat/corn-based diet (Inotiv 2920x).

BACKGROUND: Presently, we investigated hypothesized roles and mechanisms of cell type–specific, selective activation of different vascular NOX (NADPH oxidase) isoforms in obesity and metabolic syndrome. Methods: Wild-type, NOX1/2/4 global knockout mice, endothelial/VSMC-specific NOX1 knockout mice, or vascular endothelial-speicifc NOX1 knockin mice were exposed to high-fat diet feeding prior to phenotypical analyses of obesity and metabolic syndrome, as well as of exercise capacity, skeletal muscle mitochondrial function, and novel genetic signatures. Results: Expression of NOX1 was significantly upregulated in wild-type mice fed a high-fat diet. Global knockout of NOX1 (NOX1 ^−/y ), rather than of NOX2/NOX4, markedly abrogated high-fat feeding–induced body weight/fat mass gain, preadipocyte differentiation, fatty liver, glucose intolerance, and insulin/leptin resistance. Intriguingly, endothelial-specific NOX1 knockout (Cdh5cre [cadherin 5 (vascular endothelial cadherin) promoter–driven Cre recombinase (endothelial-specific Cre expression)]-NOX1CKO), rather than vascular smooth muscle-specific NOX1 knockout (Myh11cre [myosin heavy chain 11 promoter–driven Cre recombinase (smooth muscle–specific Cre expression)]-NOX1CKO), substantially alleviated obesity and metabolic syndrome. Consistently, endothelial-specific NOX1 knockin mice (Cdh5cre-NOX1CKI) fed a high-fat diet displayed exaggerated metabolic disorders. Endothelial cell–specific knockout/knockin of NOX1 was confirmed using endothelial cell washout experiments. Food/water intakes were not different from corresponding controls in high-fat fed NOX1 ^−/y , Cdh5cre-NOX1CKO, or Cdh5cre-NOX1CKI mice, indicating no difference in energy intake. Instead, spontaneous activity, exercise capacity, mitochondrial oxygen consumption/ATP production, skeletal muscle mitochondrial function (ROS production and swelling activity), and mitochondrial cristae structure were all substantially improved in NOX1 ^−/y or Cdh5cre-NOX1CKO mice, indicating augmented energy expenditure attributed to preserved skeletal muscle mitochondrial function. Supportively, Cdh5cre-NOX1CKI mice displayed deteriorated exercise capacity and skeletal muscle mitochondrial dysfunction. Endothelium-dependent vasorelaxation was restored in high-fat fed NOX1 ^−/y or Cdh5cre-NOX1CKO mice, confirming improved endothelial function. RNA-sequencing identified 4 genes ( Cntnap4 [contactin-associated protein-like 4], Sgsm1 , Tll2 , and Syt9 ) and 7 genes ( Odf3l2 , Col9a1 [collagen type IX alpha 1 chain], Cldn23 , Atp5g2 , Nkx6-3 , Ntsr2 , and Zfp69 ) significantly downregulated/upregulated in high-fat fed Cdh5cre-NOX1CKO mice, among which Cntnap4 and Col9a1 linked to muscular disorders. Importantly, we observed marked upregulation of NOX1 in isolated coronary arteries from human patients with obesity. CONCLUSIONS: Taken together, our data for the first time establish a novel and paradigm-shifting concept that endothelial NOX1 drives systematic metabolic phenotypes, via impairment in skeletal muscle mitochondrial dysfunction with novel genetic signatures. Tissue-specific targeting of endothelial NOX1 and novel candidate genes may prove to be robustly effective in treating obesity and metabolic syndrome.

The endothelium plays a central role in maintaining vascular homeostasis by orchestrating vascular tone, inflammation, healing, permeability, and thrombosis. Assessing endothelial function in vascular tissue is essential for understanding the cellular and molecular mechanisms underlying cardiovascular physiology and pathology. Traditional approaches, such as wire and pressure myography, have been instrumental in defining endothelium-dependent responses and identifying key pharmacological targets. However, the complexity and heterogeneity of endothelial cells across vascular beds and their dynamic phenotypic changes in health and disease necessitate the incorporation of new investigative strategies. Emerging methodologies, including bulk and single-cell transcriptomics, proteomics, and advanced imaging, now provide unprecedented insights into endothelial cell diversity and function. A team of leading experts in the field, who collectively reached a consensus on the most widely used techniques to evaluate endothelial function, developed these guidelines. The document establishes best practices for assessing endothelial function, from endothelial cell cultures to isolated vascular tissues, integrating conventional functional assays with modern molecular approaches. By fostering methodological consistency and embracing innovation, our goal is to enhance rigor, reproducibility, understanding, and discovery in endothelial biology.

By 2050, roughly 60% of the population will have cardiovascular disease. While a substantial amount of data has been generated over the last few decades that has aided in our understanding of cardiovascular disease pathology, less is known about how to increase resiliency to cardiovascular risk factors that individuals are exposed to on a daily basis. The vascular endothelium is considered the first line of defence against circulating noxious stimuli and, when dysfunctional, is an early risk factor for the development of cardiovascular disease. A vast amount of data has been generated demonstrating how external stress impairs the vascular endothelium; however, there is a paucity of knowledge regarding how to amplify protective pathways and ward off stress and the development of disease, which is the focus of this review. Targeting known protective endothelial pathways may be feasible to increase resiliency to vascular stress. Leveraging stress to boost defence mechanisms within the vascular endothelium is also proposed and may help identify novel therapeutic targets to protect individuals from the stress of everyday life.

Ceramides, a group of biologically active sphingolipids, have been described as the new cholesterol given strong evidence linking high plasma ceramide with endothelial damage, risk for early adverse cardiovascular events, and development of cardiometabolic disease. This relationship has sparked great interest in investigating therapeutic targets with the goal of suppressing ceramide formation. However, the growing data challenge this paradigm of ceramide as solely eliciting detrimental effects to the cardiovascular system. Studies show that ceramides are necessary for maintaining proper endothelial redox states, mechanosensation, and membrane integrity. Recent work in preclinical models and isolated human microvessels highlights that the loss of ceramide formation can in fact propagate vascular endothelial dysfunction. Here, we delve into these conflicting findings to evaluate how ceramide may be capable of exerting both beneficial and damaging effects within the vascular endothelium. We propose a unifying theory that while basal levels of ceramide in response to physiological stimuli are required for the production of vasoprotective metabolites such as S1P (sphingosine-1-phosphate), the chronic accumulation of ceramide can promote activation of pro-oxidative stress pathways in endothelial cells. Clinically, the evidence discussed here highlights the potential challenges associated with therapeutic suppression of ceramide formation as a means of reducing cardiovascular disease risk.