Anne-Christine Peyter

Background: Regulation of the human umbilical circulation under physiological and pathological conditions remains poorly understood. We previously demonstrated that intrauterine growth restriction (IUGR) is associated with sex-specific alterations in the human umbilical circulation. Our data strongly suggest a differential contribution of subcellular compartmentation depending on fetal sex, vessel type and the presence of IUGR. We therefore developed a protocol to isolate and culture umbilical vascular cells to further investigate the relative contribution of each cell type and subcellular compartmentation to the human umbilical circulation regulation. Methods and results: Human umbilical cords and cord blood were collected just after delivery. Mononuclear cells were recovered from cord blood using a Ficoll gradient and cultured to obtain endothelial colony-forming cells (ECFCs). Endothelial cells (ECs) were isolated from human umbilical vein (HUV) and arteries (HUAs) by collagenase/dispase digestion, and vascular smooth muscle cells (SMCs) by migration from vascular explants. All cell types were characterized by visualization, and by analysis of biomarkers using immunocytofluorescence and Western blot. ECFCs were also submitted to polychromatic flow cytometry analysis. Conclusions: This protocol enables simultaneous isolation and culture of ECFCs, HUVECs, HUAECs, HUVSMCs and HUASMCs from the same umbilical cord. It is simpler, faster and more cost-effective than other previously published methods, with good success rates. This will be helpful to further investigate the regulatory mechanisms implicated in the human umbilical circulation under physiological and pathological conditions and to study the influence of fetal sex.

Adverse events during the perinatal period are associated with an increased risk to develop cardiometabolic diseases later in life. We established a murine model to study long-term effects of perinatal hypoxia (PH) on the pulmonary circulation. We previously demonstrated that PH led to an impaired regulation of pulmonary vascular tone in adulthood, linked to alterations in K+ channels in males and in the nitric oxide (NO)/cyclic guanosine monophosphate pathway in females. Moreover, simultaneous administration of inhaled NO (iNO) during PH exposure prevented adverse effects of PH on adult pulmonary vasculature in females. The present study showed that PH induced a significant increase in right ventricular pressure in males and females, and an enhanced sensitivity to acute hypoxia in females. PH significantly reduced acetylcholine-induced relaxation in pulmonary artery, to a greater extent in females than in males. PH led to right ventricular hypertrophy in adulthood, appearing earlier in males than in females. Morphometric measurements showed a significant increase in the number of 25-75-µm pulmonary vessels in male lungs following PH, probably resulting in increased pulmonary vascular resistance. The effects of prolonged hypoxia in adulthood differed between males and females. Perinatal iNO during PH prevented PH-induced alterations in the cardiopulmonary system, whereas perinatal iNO alone could have some adverse effects. Therefore, PH led to long-lasting alterations in the regulation of adult pulmonary circulation, which vary between males and females. In males, the increased pulmonary vascular resistance was associated with morphological changes besides functional alterations, whereas females showed an important pulmonary vascular dysfunction.

Fetal growth restriction (FGR) is a common complication of pregnancy, associated with major perinatal mortality and morbidity, and with an increased risk to develop cardiometabolic diseases later in life. There is currently no effective approach to prevent or treat FGR, despite numerous animal and human studies assessing substances likely to improve fetal growth. Phosphodiesterase (PDE) inhibitors appeared as promising drugs to improve FGR management. However, to date, studies have led to somewhat disappointing or controversial results. In this Opinion article, we would like to draw attention to the need to consider the biological sex and the relative reactivity of human umbilical vein and arteries when developing therapeutic interventions to improve human umbilical circulation using PDE inhibitors. Indeed, we suspect that fetal sex, vessel type and the presence of FGR may influence subcellular compartmentation, which could jeopardize beneficial effects of PDE inhibitors.

In humans, glucocorticoid resistance is attributed to mutations in the glucocorticoid receptor (GR). Most of these mutations result in decreased ligand binding, transactivation, and/or translocation, albeit with normal protein abundances. However, there is no clear genotype‒phenotype relationship between the severity or age at disease presentation and the degree of functional loss of the receptor. Previously, we documented that a GR^+/− rat line developed clinical features of glucocorticoid resistance, namely, hypercortisolemia, adrenal hyperplasia, and salt-sensitive hypertension. In this study, we analyzed the GR^+/em4 rat model heterozygously mutant for the deletion of exon 3, which encompasses the second zinc finger, including the domains of DNA binding, dimerization, and nuclear localization signals. On a standard diet, mutant rats exhibited a trend toward increased corticosterone levels and a normal systolic blood pressure and heart rate but presented with adrenal hyperplasia. They exhibited increased adrenal soluble epoxide hydroxylase (sEH), favoring an increase in less active polyunsaturated fatty acids. Indeed, a significant increase in nonactive omega-3 and omega-6 polyunsaturated fatty acids, such as 5(6)-DiHETrE or 9(10)-DiHOME, was observed with advanced age (10 versus 5 weeks old) and following a switch to a high-salt diet accompanied by salt-sensitive hypertension. In thoracic aortas, a reduced soluble epoxide hydrolase (sEH) protein abundance resulted in altered vascular reactivity upon a standard diet, which was blunted upon a high-salt diet. In conclusion, mutations in the GR affecting the ligand-binding domain as well as the dimerization domain resulted in deregulated GR signaling, favoring salt-sensitive hypertension in the absence of obvious mineralocorticoid excess.

The effects of phosphodiesterase inhibition on the umbilical circulation depend on the presence of intrauterine growth restriction, the fetal sex, vessel type, and vasoconstrictors implicated. The human umbilical vascular tone regulation is complex and depends on the amount and activity of specific proteins but also probably on the subcellular organization mediating protein interactions. Therefore, therapeutic interventions using phosphodiesterase inhibitors to improve the placental-fetal circulation should consider fetal sex and both umbilical vein and artery reactivity.

Introduction Lower extremity peripheral artery disease (PAD), due to narrowing of the arteries supplying the legs, represents a major cause of physical disability. Exercise training is an important part of PAD treatment, improving functional capacity. Recent research suggests that exercise training-induced endothelial function improvement is involved in the benefits of exercise in PAD (Lee et al., 2023); however, the underlying molecular mechanisms remain unknown. In addition, the potential vascular benefits of high intensity training in PAD remain unexplored. Therefore, the aim of the study was to compare the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on endurance capacity, vascular endothelial function and expression of genes related to nitric oxide (NO) bioavailability in a mouse model of PAD. Methods PAD disease model was generated in male C57BL/6 mice by ligation of the right iliac artery. Mice with PAD were divided into 3 groups: sedentary control group (SED), MICT (40 min running at 70% of maximal aerobic speed) and HIIT (8 intervals of 2.5 minutes running at 90% of maximal aerobic speed combined with 2.5 minutes of running at 50% of maximal aerobic speed). Mice were exercised three times per week for 8 weeks. Maximal running time was determined by an incremental test until exhaustion. Vascular reactivity was assessed by measuring vasoconstrictor responses to phenylephrine as well as endothelium-dependent and -independent vasodilator responses to acetylcholine and DEA-NONOate, respectively, in isolated aortic rings. Gene expression of antioxidant enzymes (catalase and superoxide dismutase isoforms), prooxidant NADPH oxidase subunits (p22phox and p47phox), enzyme involved in NO production (endothelial NO synthase), vascular smooth muscle cell relaxation (soluble guanylate cyclase) and receptors involved in vascular tone regulation (a₁ and b₂ adrenergic receptors) was determined in aortic tissue by qPCR. Results Maximal running time was improved in both MICT and HIIT as compared to SED at the end of the study, but with no significant difference between the two exercise groups. Phenylephrine-induced vasoconstriction as well as vasodilator responses to acetylcholine and DEA-NONOate were significantly improved in MICT compared to SED while no significant differences were reported between HIIT and SED. There was no significant difference in gene expression among the three groups. Discussion/Conclusion MICT and HIIT were equally effective at improving endurance capacity in mice with PAD. However, only MICT improved vascular reactivity. References Lee, J., Zarezadehmehrizi, A., LaVoy, E., Markofski, M., & Park, Y. (2023). Exercise training improves brachial artery endothelial function, but does not alter inflammatory biomarkers in patients with peripheral artery disease: A systematic review and meta-analysis. Journal of Cardiovascular Translational Research. Advance online publication. https://doi.org/10.1007/s12265-023-10451-0

Individuals born after intrauterine growth restriction (IUGR) are at risk of developing cardiovascular diseases (CVDs). Endothelial dysfunction plays a role in the pathogenesis of CVDs; and endothelial colony-forming cells (ECFCs) have been identified as key factors in endothelial repair. In a rat model of IUGR induced by a maternal low-protein diet, we observed an altered functionality of ECFCs in 6-month-old males, which was associated with arterial hypertension related to oxidative stress and stress-induced premature senescence (SIPS). Resveratrol (R), a polyphenol compound, was found to improve cardiovascular function. In this study, we investigated whether resveratrol could reverse ECFC dysfunctions in the IUGR group. ECFCs were isolated from IUGR and control (CTRL) males and were treated with R (1 μM) or dimethylsulfoxide (DMSO) for 48 h. In the IUGR-ECFCs, R increased proliferation (5′-bromo-2′-deoxyuridine (BrdU) incorporation, p < 0.001) and improved capillary-like outgrowth sprout formation (in Matrigel), nitric oxide (NO) production (fluorescent dye, p < 0.01), and endothelial nitric oxide synthase (eNOS) expression (immunofluorescence, p < 0.001). In addition, R decreased oxidative stress with reduced superoxide anion production (fluorescent dye, p < 0.001); increased Cu/Zn superoxide dismutase expression (Western blot, p < 0.05); and reversed SIPS with decreased beta-galactosidase activity (p < 0.001), and decreased p16^ink4a (p < 0.05) and increased Sirtuin-1 (p < 0.05) expressions (Western blot). No effects of R were observed in the CTRL-ECFCs. These results suggest that R reverses long-term ECFC dysfunctions related to IUGR.

Metabolic syndrome (MetS) refers to cardiometabolic risk factors, such as visceral obesity, dyslipidemia, hyperglycemia/insulin resistance, arterial hypertension and non-alcoholic fatty liver disease (NAFLD). Individuals born after intrauterine growth restriction (IUGR) are particularly at risk of developing metabolic/hepatic disorders later in life. Oxidative stress and cellular senescence have been associated with MetS and are observed in infants born following IUGR. However, whether these mechanisms could be particularly associated with the development of NAFLD in these individuals is still unknown. IUGR was induced in rats by a maternal low-protein diet during gestation versus. a control (CTRL) diet. In six-month-old offspring, we observed an increased visceral fat mass, glucose intolerance, and hepatic alterations (increased transaminase levels, triglyceride and neutral lipid deposit) in male rats with induced IUGR compared with the CTRL males; no differences were found in females. In IUGR male livers, we identified some markers of stress-induced premature senescence (SIPS) (lipofuscin deposit, increased protein expression of p21^WAF, p16^INK4a and Acp53, but decreased pRb/Rb ratio, foxo-1 and sirtuin-1 protein and mRNA expression) associated with oxidative stress (higher superoxide anion levels, DNA damages, decreased Cu/Zn SOD, increased catalase protein expression, increased nfe2 and decreased keap1 mRNA expression). Impaired lipogenesis pathways (decreased pAMPK/AMPK ratio, increased pAKT/AKT ratio, SREBP1 and PPARγ protein expression) were also observed in IUGR male livers. At birth, no differences were observed in liver histology, markers of SIPS and oxidative stress between CTRL and IUGR males. These data demonstrate that the livers of IUGR males at adulthood display SIPS and impaired liver structure and function related to oxidative stress and allow the identification of specific therapeutic strategies to limit or prevent adverse consequences of IUGR, particularly metabolic and hepatic disorders.

Infants born after intrauterine growth restriction (IUGR) are at risk of developing arterial hypertension at adulthood. The endothelium plays a major role in the pathogenesis of hypertension. Endothelial colony-forming cells (ECFCs), critical circulating components of the endothelium, are involved in vasculo-and angiogenesis and in endothelium repair. We previously described impaired functionality of ECFCs in cord blood of low-birth-weight newborns. However, whether early ECFC alterations persist thereafter and could be associated with hypertension in individuals born after IUGR remains unknown. A rat model of IUGR was induced by a maternal low-protein diet during gestation versus a control (CTRL) diet. In six-month-old offspring, only IUGR males have increased systolic blood pressure (tail-cuff plethysmography) and microvascular rarefaction (immunofluorescence). ECFCs isolated from bone marrow of IUGR versus CTRL males displayed a decreased proportion of CD31+ versus CD146+ staining on CD45− cells, CD34 expression (flow cytometry, immunofluorescence), reduced proliferation (BrdU incorporation), and an impaired capacity to form capillary-like structures (Matrigel test), associated with an impaired angiogenic profile (immunofluorescence). These dysfunctions were associated with oxidative stress (increased superoxide anion levels (fluorescent dye), decreased superoxide dismutase protein expression, increased DNA damage (immunofluorescence), and stress-induced premature senescence (SIPS; increased beta-galactosidase activity, increased p16^INK4a, and decreased sirtuin-1 protein expression). This study demonstrated an impaired functionality of ECFCs at adulthood associated with arterial hypertension in individuals born after IUGR.

Metabolic syndrome (MetS) is a cluster of several disorders, such as hypertension, central obesity, dyslipidemia, hyperglycemia, insulin resistance and non-alcoholic fatty liver disease. Despite health policies based on the promotion of physical exercise, the reduction of calorie intake and the consumption of healthy food, there is still a global rise in the incidence and prevalence of MetS in the world. This phenomenon can partly be explained by the fact that adverse events in the perinatal period can increase the susceptibility to develop cardiometabolic diseases in adulthood. Individuals born after intrauterine growth restriction (IUGR) are particularly at risk of developing cardiovascular diseases (CVD) and metabolic disorders later in life. It has been shown that alterations in the structural and functional integrity of the endothelium can lead to the development of cardiometabolic diseases. The endothelial progenitor cells (EPCs) are circulating components of the endothelium playing a major role in vascular homeostasis. An association has been found between the maintenance of endothelial structure and function by EPCs and their ability to differentiate and repair damaged endothelial tissue. In this narrative review, we explore the alterations of EPCs observed in individuals with cardiometabolic disorders, describe some mechanisms related to such dysfunction and propose some therapeutical approaches to reverse the EPCs dysfunction.