What’s Known

In animal models, fetal alcohol exposure was found to cause various heart and vascular abnormalities, including heart cell proliferation, increased arterial stiffness, and impaired vascular vasodilation function. Furthermore, studies found that long-term ethanol exposure in pregnant rats resulted in an increase in mean arterial pressure in offspring at six months of age.

What’s New

Maternal ethanol exposure causes aorta wall remodeling on post-natal days 21 and 90. Ethanol causes an increase in inflammatory mediators in aorta tissue, as well as a change in hemodynamic variables, on post-natal days 21 and 90.

Introduction

Alcohol consumption during pregnancy is associated with a number of fatal adverse effects, including premature birth, intrauterine growth restriction, craniofacial deficits, and central nervous system dysfunction, collectively named fetal alcohol spectrum disorders (FASD). ^{1 , 2} As the neurodevelopmental disorders induced by maternal alcohol consumption are visible and have obvious manifestations, early studies on FASD in terms of neurodevelopmental and craniofacial deficits have received much attention. Over the last two decades, researchers have focused on the impact of maternal ethanol consumption on cardiovascular system abnormalities, particularly in response to binge drinking. ^{3 , 4} Fetal alcohol exposure was found to induce some measures of heart and vascular abnormalities such as heart cell proliferation, increased arterial stiffening, and altered vascular vasodilation function. ^{4 , 5} Turcott and others also reported that long-term ethanol exposure in pregnant rats increased the mean arterial pressure and impaired the aortic endothelium-dependent relaxation in the offspring at six months of age. ⁶ Cardiovascular malformation and vascular stiffness have also been reported in children exposed to ethanol during the gestation period. ^{7 , 8} Our laboratory recently found that ethanol exposure during pregnancy and lactation caused cardiac structural and biochemical changes, hippocampus defects, and testis abnormalities in the rats’ offspring 21 and 90 days after birth. ^{5 , 9 , 10} These studies provide evidence that chronic ethanol exposure during cardiovascular system development predisposes the adult offspring to a risk of cardiovascular dysfunction.

The purpose of this study was to explore the effect of maternal ethanol ingestion on the aorta of male rat offspring in terms of functional histological and molecular endpoints at days 21 and 90 after birth.

Materials and Methods

Experimental Procedure

This experimental study was conducted at Urmia University of Medical Sciences (Urmia, Iran) in 2019. The study protocol was approved by the Ethics Committee of Urmia University of Medical Sciences (code: IR.UMSU.RES.1398.129). The animal care and experimental procedures were performed according to the national guidelines, in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Twenty pregnant date-mated rats were housed separately and assigned to two groups: alcohol and control. ⁵ From Gestation Day (GD) seven to postnatal day (PN) 21(lactation period), the rats in the alcohol group were given ethanol at a dose of 4.5 g/Kg body weight (Merck KGaA, Darmstadt, Germany) diluted with distilled water (20% w/v) intragastrically by gavage once a day. Due to the impact of sex differences on response to ethanol, only male pups were used in this study. During the weaning period, the pups (control and ethanol-exposed) had free access to nipples. Ketamine (Alfasan, Holland; 10%, 80 mg/Kg B.W, IP) and xylazine (Alfasan, Holland; 2%, 10 mg/Kg B.W, IP) were used to anesthetize the offspring from each group on PN21 and PN90 (n=8 offspring from each group PN21 and PN90).

Blood Pressure and Hypertension

Systemic hemodynamic variables and heart rates were determined using a physiograph and a digital waveform contour analysis method directly recorded from the carotid artery (NARCO, Bio-system, USA). The detail of the protocol were outlined in our previous work. ¹¹ Power Lab Software (ADInstruments, Australia) was used to analyze the data, i.e., the recorded digital volume pulse.

Tissue Sampling

After blood pressure measurement (only in PN90 pups), the thoracic cavity was opened, and the aorta was dissected from the root to the abdomen descending section. The harvested aorta was washed and cleansed to remove any adventitial tissues, fat, and blood clots.

For histological analysis, formalin was used to fix aorta samples. After the dehydration steps, the samples were embedded in paraffin and sectioned at 5 µm for histological evaluation. In order to assess the histopathological alterations, eight slides per group and four non-overlapping fields of view per section from two to three sections per animal were analyzed.

For biochemical analysis, the remaining half of the aorta was rinsed with ice-cold normal saline and wiped with filter papers for biochemical analysis. After, mixing with an ice-cold extraction buffer (10% wt/Vol) including a 50 mM phosphate buffer (pH 7.4), Ultra Turrax (T10B, IKA, Germany) was used for homogenizing the samples. The products were then centrifuged at 10,000 ×g at 4 °C for 20 minutes, and the supernatant was frozen at -80 °C for further analysis.

Histopathological Assessment

Sections of the aorta were stained with Masson trichrome according to the manufacturer’s instruction (Asiapajohesh, Amol, Iran). The intensity of aorta fibrosis in different groups was assessed using a semi-quantitative method described by Ashcroft and others. ¹² They applied criteria for scoring fibrosis intensity as 0=normal aorta tunica media, 1=minimal fibrosis thickening of the aorta tunica media, 2 and 3=moderate thickening of aorta tunica media without obvious damage to the structure of the aorta tissue, 4 and 5=increased fibrosis with definite damage to the architecture of the aorta and formation of fibrosis bands or small fibrosis masses, 6 and 7=severe distortion of the structure and large fibrosis areas, and 8=total fibrotic obliteration.

After preparing the tissue sections, Verhoeff’s van Gieson and Periodic Acid-Schiff staining were used to detect elastin bands. To assess the atherosclerotic features, eight sections of the aorta from different experimental groups were used. Herein, the total aorta thickness, tunica media, tunica adventitia, smooth muscle cell layers’ width, and elastic fiber thickness were measured using a linear scale-ocular micrometer with a 2.5 mm interval between divisions under x400 magnification with a digital camera equipped with a research microscope (Olympus, CX 31, Japan).

Biochemical Examinations

The levels of TNF-α, ICAM-1, NF-κB, and endothelin-1 in the aorta tissue were analyzed using the quantitative sandwich enzyme immunoassay method, commercial rat TNF-α, ICAM-1, endothelin-1, and NF-κB (Shanghaicrystal Day, Biotech, China) kits, according to the manufacturer’s instruction.

Statistical Analysis

The normal distribution of the data within each group was verified through the Kolmogorov–Smirnov test using SPSS software (version 17.0; SPSS Inc, Chicago, IL, USA). In addition, the statistical differences between the control and ethanol groups were tested through an independent samples t test. The data obtained from each test were presented as mean±SEM, and P<0.05 was considered to be statistically significant.

Results

Aorta Wall Cytokines and Chemokine Changes

The changes in inflammatory cytokines and chemokine levels in the offspring of the experimental groups are presented in table 1. Pre- and early postnatal animals exposed to ethanol showed significantly higher levels of pup aorta tissue, TNF-α, and NF-κB on both PN21 and PN 90, when compared with control pups of the same age groups (P<0.001). As compared to the control group, ICAM-1 level in the ethanol-treated groups showed a significant increase in the aorta wall of liters in both PN21 and PN90 (P<0.001). On PN90, the Endothelin-1 level was significantly higher in the aorta tissue of pups from the ethanol group than in the control group (P<0.001).

Aorta Wall Thickness Changes

The data regarding the aorta wall elastin layers’ changes in litters is summarized in table 2 and figure 1. As compared to the control pups of the same age groups, the total aorta wall thickness in dams exposed to ethanol increased significantly on both PN21 and PN90 (P<0.001). On PN21, tunica media and tunica adventitia thickness increased significantly in the aorta of the ethanol group compared to the control group (P<0.001). On PN90, tunica media showed a significant increase compared to the control group (P<0.001), but there were no significant differences regarding the tunica adventitia thickness changes between the ethanol and control groups (P=0.37). The elastin fiber thickness and entire elastin thickness were significantly increased in the aorta of the ethanol-treated litters compared to the control group on both PN21 and PN90, P=0.044, and P=0.015, respectively. On PN90, the ethanol group had a significantly higher elastin fiber interval than the control group (P=0.014). There was no significant difference between the ethanol and control groups in the elastin fiber interval (P=0.55), on PN21. The smooth muscle cells thickness on PN90 was significantly greater than in the control group (P<0.001). In the aorta of the ethanol-treated group, the elastin/media ratio was significantly higher than the control group on PN21 (P=0.011). On PN90, there were no significant differences in the elastin/media ratio between the ethanol and control groups (P=0.31). On PN21 and PN90, the media/aorta ratio in ethanol-exposed groups was significantly higher than that of the control groups (P<0.001).

Figure 1. The ethanol-treated group showed an increase in total aorta thickness, tunica media, tunica adventitia, elastin fiber thickness, fiber interval, and elastin/media ratio compared to the controls on PN21 and PN90 (Verhoeff’s van Gieson). Arrows show elastin layer thickness in different groups (×400).

Aorta Wall Collagen Changes

Masson’s trichrome staining results revealed no lesion score in the aorta media of the control group on PN21 and PN90, as well as the ethanol group on PN21 (grade=0). On PN90, the microscopic lesion scores in the tunica media of the aorta were 2 and 3, indicating moderate thickening of aorta tunica media without obvious damage to the aorta tissue structure. Tunica adventitia of the aorta was normally composed of collagen and fibrosis, which was visible in both the control and ethanol-treated groups. Ethanol-treated groups indicated a significant increase in fibrosis layer thickness on both PN21 and PN90, compared to the controls (figure 2).

Figure 2. The arrows show the collagen accumulation in the intima region of the PN 90 ethanol treated rats, which appeared in blue color (Masson trichrome, ×400).

Blood Pressure

The digital waveform contour analysis method was used to analyze systemic hemodynamic variables and heart rates in PN90 pups, and the results are reported in table 3. Heart rates did not differ significantly between the ethanol-treated and control groups (P=0.67). On PN90, the ethanol-treated pups had significantly higher systolic, diastolic, and mean arterial blood pressures than the control pups (P<0.001). When compared to the control group, dicrotic notch pressure and pulse pressure were increased significantly in the ethanol group (P=0.05).

Discussion

The findings of the present study provide several lines of evidence such as increased aorta wall thickness, high blood pressure, and increased inflammatory cytokines, including TNF-α, ICAM-1, NF-κB, and endothelin levels in the aorta tissue of the offspring exposed to ethanol during pregnancy and early postnatal period 90 days after birth. All the measurements performed herein were known as possible risk factors for cardiovascular abnormalities, which also predispose the aorta wall to atherosclerosis. Previous studies on the effect of ethanol feeding during pregnancy on offspring blood pressure variables reported ambiguous results. Several studies found that mothers’ ethanol exposure during the gestation period led to a significant increase in systolic, diastolic, and mean arterial pressure in their offspring, which is consistent with the findings of the current study. ^{6 , 13 , 14} Other studies on animal models or on offspring of mothers who consumed alcohol during pregnancy found that ethanol consumption during gestation had no effect on blood pressure variables. ^{8 , 15} The discrepancy between these studies and our findings could be attributed to technical differences in blood pressure measurement methods. Previous studies obtained blood pressure-related variables using tail-cuff methods in animal studies and brachial artery-cuff methods in human studies, while we employed contour analysis of hemodynamic variables using carotid arterial cannulation. Although this method is aggressive, the results are more informative and reliable than traditional methods such as tail-cuff methods. ^{16 , 17}

In our study, in addition to a significant rise in systolic, diastolic, and mean arterial pressure, there was a significant increase in dicrotic or reflected pressure in pups obtained from the ethanol group 90 days after birth. The height of the dichroic notch indicates reduced nitric oxide (NO) production, which leads to aorta wall stiffness. It acts as a strong vasodilator contributing to vessel diameters, tone, resistance, and compliance of vasculature. ¹⁸ Two reviews of pulse waves in human fingers and rabbit auricular arteries revealed that changes in the NO pathway were associated with variations in the relative height of the dichroic notch. ^{18 , 19}

In addition to an increase in dicrotic pressure, the current study found a parallel significant increase in total aorta thickness, tunica media thickness, elastin fiber thickness, elastin fiber interval, and the media/aorta ratio in the ethanol group compared to the control animals. We also found significant collagen deposition (grade 2, 3) in the elastin fiber interval and a fibrosis score of grade 4-5 in the adventitia of the ethanol group, indicating severe adventitia thickening. It has been reported that elevated collagen deposition and elastin fraction in the artery wall increased arterial stiffness. ²⁰

Recently, arterial stiffness has been proposed as an independent predictor of cardiovascular diseases, such as increased pulse pressure/hypertension, left ventricular dysfunction/hypertrophy, and myocardial ischemia/increased metabolic demands. ^{20 - 22}

It’s worth noting that aortic media thickens is responsible for better tension distribution across the aortic wall, and thus its thickness is considered an index of generalized atherosclerosis. ^{23 , 24}

From a histological viewpoint, the media is formed by elastin fibers, and the fiber interval mainly includes smooth muscle cells and extracellular matrix proteins. In contrast to the control group, both of these components were significantly elevated in the ethanol group. Previously, ethanol consumption during gestation-induced cardiovascular anomalies was shown to prevent normal cardiogenesis in FAS animal models through various molecular mechanisms, including retinoic acid signaling pathway, sonic hedgehog signaling, and Wnt/β-catenin signaling pathway. ^{25 - 27}

While acknowledging the previous works, we proposed in this study that inflammatory cytokines, which are key molecular mediators in atherosclerosis, may contribute to ethanol-induced aorta abnormalities in pups. Hence, since atherosclerosis is now recognized as a chronic inflammatory condition, alcohol consumption is clearly associated with a higher risk of atherosclerosis. On both PN21 and PN90, the level of NF-κB in the ethanol-treated groups was significantly higher than in the control groups. The transcriptional factor of the nuclear factor kappa B signaling pathway in the vascular endothelial cells is responsible for numerous gene expressions involved in inflammation, immune responses, cell growth, and adhesion molecules such as ICAM-1, vascular adhesion molecule (VCAM)-1, TNF-α, IL-6, and beta-selectin on the cell surface. ^{28 , 29} In addition, numerous genes expressed by the NF-κB pathway may be related to atherosclerosis plaque formation.

TNF-α has also been shown to directly activate NF-κB, inducing an inflammatory response. ³⁰ Although TNF-α elevation is secondary to NF-κB or vice versa, our study found a significant increase in TNF-α and ICAM-1 in the aorta tissue of litters. Numerous studies have shown that TNF-α promoted the generation of reactive oxygen species (ROS) in the endothelial cell through the activation of NADPH oxidase. ^{30 - 32}

ROS generation and the subsequent TNF-activation seem to act as a second messenger, regulating the expression of adhesive molecules such as VCAM-1 and ICAM-1. ³² Moreover, TNF-α, as a putative inflammatory cytokine, may contribute to the development and progression of atherosclerosis by facilitating the transcytosis of low density lipoprotein (LDL) across endothelial cells and the retention of LDL in the vascular wall. ³³ In the current study, we found that ethanol significantly increased endothelin-1 level in the aorta tissue of rats. Endothelin-1 is primarily secreted by endothelial cells and plays a major role in the maintenance of basal vascular tone and vessel constriction. In addition, a study reported that endothelin-1 had mitogenic properties on vascular smooth muscle cells and stimulated the proliferation of coronary vascular smooth muscle cells. This effect of endothelin-1 has been observed in atherosclerotic coronary plaques and may be associated with atherosclerosis. ³⁴

The current research had several limitations. Although evidence of LV dysfunction, such as decreased ejection fraction, is fairly straightforward to detect in rodents by echocardiography, we didn’t use echocardiography in this study. This study focused only on the phenotypic effects of maternal alcohol consumption on the aorta of the offspring, with little attention paid to the mechanisms that could explain the phenotype.

Conclusion

Taking these findings together, we concluded that daily maternal alcohol consumption during gestation and in the early postnatal period induced aorta wall remodeling in the offspring, which manifested itself as increased aorta wall stiffness and hypertension even 90 days after birth. In addition, the elevation of vascular biomarkers of inflammation and atherosclerosis revealed a more significant and stronger association between maternal ethanol consumption-induced aorta structure changes through inflammatory stress. These findings suggest that maladaptive alterations in the fetal aorta in response to ethanol exposure during gestation may persist postnatally and increase the risk of cardiovascular abnormalities in adulthood.

Acknowledgement

This work was supported by the Research Administration of Urmia University of Medical Sciences, Urmia, Iran, and the authors are grateful to the UMSU Research Administration for their cooperation.

Authors’ Contribution

H.S and A.S: Contributed to conception, design; S.S, F.N.M, R.N, and Y.R: Contributed to data analysis; Y.R: Contributed to critically revising the work; H.S, A.S, S.S, F.N.M and R.N: Contributed to drafting the manuscript. All authors approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflict of Interest

None declared.

References

1. Kesmodel U, Olsen SF, Secher NJ. Does alcohol increase the risk of preterm delivery?. Epidemiology. 2000; 11:512-8. DOI | PubMed
2. Sokol RJ, Delaney-Black V, Nordstrom B. Fetal alcohol spectrum disorder. JAMA. 2003; 290:2996-9. DOI | PubMed
3. Pruett D, Waterman EH, Caughey AB. Fetal alcohol exposure: consequences, diagnosis, and treatment. Obstet Gynecol Surv. 2013; 68:62-9. DOI | PubMed
4. Parkington HC, Kenna KR, Sozo F, Coleman HA, Bocking A, Brien JF, et al. Maternal alcohol consumption in pregnancy enhances arterial stiffness and alters vasodilator function that varies between vascular beds in fetal sheep. J Physiol. 2014; 592:2591-603. Publisher Full Text | DOI | PubMed
5. Shirpoor A, Nemati S, Ansari MH, Ilkhanizadeh B. The protective effect of vitamin E against prenatal and early postnatal ethanol treatment-induced heart abnormality in rats: a 3-month follow-up study. Int Immunopharmacol. 2015; 26:72-9. DOI | PubMed
6. Turcotte LA, Aberle NS, Norby FL, Wang GJ, Ren J. Influence of prenatal ethanol exposure on vascular contractile response in rat thoracic aorta. Alcohol. 2002; 26:75-81. DOI | PubMed
7. Kvigne VL, Leonardson GR, Neff-Smith M, Brock E, Borzelleca J, Welty TK. Characteristics of children who have full or incomplete fetal alcohol syndrome. J Pediatr. 2004; 145:635-40. DOI | PubMed
8. Morley R, Dwyer T, Hynes KL, Cochrane J, Ponsonby AL, Parkington HC, et al. Maternal alcohol intake and offspring pulse wave velocity. Neonatology. 2010; 97:204-11. Publisher Full Text | DOI | PubMed
9. Shirpoor A, Salami S, Khadem-Ansari MH, Minassian S, Yegiazarian M. Protective effect of vitamin E against ethanol-induced hyperhomocysteinemia, DNA damage, and atrophy in the developing male rat brain. Alcohol Clin Exp Res. 2009; 33:1181-6. DOI | PubMed
10. Shirpoor A, Norouzi L, Khadem-Ansari MH, Ilkhanizadeh B, Karimipour M. The Protective Effect of Vitamin E on Morphological and Biochemical Alteration Induced by Pre and Postnatal Ethanol Administration in the Testis of Male Rat Offspring: A Three Months Follow-up Study. J Reprod Infertil. 2014; 15:134-41. Publisher Full Text | PubMed
11. Shirpoor A, Salami S, Khadem-Ansari MH, Heshmatian B, Ilkhanizadeh B. Long-term ethanol consumption initiates atherosclerosis in rat aorta through inflammatory stress and endothelial dysfunction. Vascul Pharmacol. 2012; 57:72-7. DOI | PubMed
12. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988; 41:467-70. Publisher Full Text | DOI | PubMed
13. Nguyen VB, Probyn ME, Campbell F, Yin KV, Samuel CS, Zimanyi MA, et al. Low-dose maternal alcohol consumption: effects in the hearts of offspring in early life and adulthood. Physiol Rep. 2014; 2Publisher Full Text | DOI | PubMed
14. Gray SP, Denton KM, Cullen-McEwen L, Bertram JF, Moritz KM. Prenatal exposure to alcohol reduces nephron number and raises blood pressure in progeny. J Am Soc Nephrol. 2010; 21:1891-902. Publisher Full Text | DOI | PubMed
15. Gray SP, Kenna K, Bertram JF, Hoy WE, Yan EB, Bocking AD, et al. Repeated ethanol exposure during late gestation decreases nephron endowment in fetal sheep. Am J Physiol Regul Integr Comp Physiol. 2008; 295:R568-74. DOI | PubMed
16. Fritz M, Rinaldi G. Blood pressure measurement with the tail-cuff method in Wistar and spontaneously hypertensive rats: influence of adrenergic- and nitric oxide-mediated vasomotion. J Pharmacol Toxicol Methods. 2008; 58:215-21. DOI | PubMed
17. Ibrahim J, Berk BC, Hughes AD. Comparison of simultaneous measurements of blood pressure by tail-cuff and carotid arterial methods in conscious spontaneously hypertensive and Wistar-Kyoto rats. Clin Exp Hypertens. 2006; 28:57-72. DOI | PubMed
18. Alastruey J, Nagel SR, Nier BA, Hunt AA, Weinberg PD, Peiro J. Modelling pulse wave propagation in the rabbit systemic circulation to assess the effects of altered nitric oxide synthesis. J Biomech. 2009; 42:2116-23. DOI | PubMed
19. Chowienczyk PJ, Kelly RP, MacCallum H, Millasseau SC, Andersson TL, Gosling RG, et al. Photoplethysmographic assessment of pulse wave reflection: blunted response to endothelium-dependent beta2-adrenergic vasodilation in type II diabetes mellitus. J Am Coll Cardiol. 1999; 34:2007-14. DOI | PubMed
20. Woodman RJ, Watts GF. Measurement and application of arterial stiffness in clinical research: focus on new methodologies and diabetes mellitus. Med Sci Monit. 2003; 9:RA81-9. PubMed
21. Laurent S, Boutouyrie P, Asmar R, Gautier I, Laloux B, Guize L, et al. Aortic stiffness is an independent predictor of all-cause and cardiovascular mortality in hypertensive patients. Hypertension. 2001; 37:1236-41. DOI | PubMed
22. Blacher J, Pannier B, Guerin AP, Marchais SJ, Safar ME, London GM. Carotid arterial stiffness as a predictor of cardiovascular and all-cause mortality in end-stage renal disease. Hypertension. 1998; 32:570-4. DOI | PubMed
23. Wolinsky H. Long-term effects of hypertension on the rat aortic wall and their relation to concurrent aging changes. Morphological and chemical studies. Circ Res. 1972; 30:301-9. DOI | PubMed
24. Demircioglu F, Kocyigit A, Arslan N, Cakmakci H, Hizli S, Sedat AT. Intima-media thickness of carotid artery and susceptibility to atherosclerosis in obese children with nonalcoholic fatty liver disease. J Pediatr Gastroenterol Nutr. 2008; 47:68-75. DOI | PubMed
25. Deltour L, Ang HL, Duester G. Ethanol inhibition of retinoic acid synthesis as a potential mechanism for fetal alcohol syndrome. FASEB J. 1996; 10:1050-7. PubMed
26. Brennan D, Giles S. Sonic hedgehog expression is disrupted following in ovo ethanol exposure during early chick eye development. Reprod Toxicol. 2013; 41:49-56. DOI | PubMed
27. Serrano M, Han M, Brinez P, Linask KK. Fetal alcohol syndrome: cardiac birth defects in mice and prevention with folate. Am J Obstet Gynecol. 2010; 203:75. DOI | PubMed
28. Jayachandran M, Chandrasekaran B, Namasivayam N. Geraniol attenuates fibrosis and exerts anti-inflammatory effects on diet induced atherogenesis by NF-kappaB signaling pathway. Eur J Pharmacol. 2015; 762:102-11. DOI | PubMed
29. Baker RG, Hayden MS, Ghosh S. NF-kappaB, inflammation, and metabolic disease. Cell Metab. 2011; 13:11-22. Publisher Full Text | DOI | PubMed
30. Zhang T, Sun L, Liu R, Zhang D, Lan X, Huang C, et al. A novel naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-kappaB activity in RAW264.7 macrophages. Mol Pharm. 2012; 9:671-7. DOI | PubMed
31. Kim SR, Bae YH, Bae SK, Choi KS, Yoon KH, Koo TH, et al. Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-kappaB activation in endothelial cells. Biochim Biophys Acta. 2008; 1783:886-95. DOI | PubMed
32. Cominacini L, Pasini A, Garbin U, Evangelista S, Crea AE, Tagliacozzi D, et al. Zofenopril inhibits the expression of adhesion molecules on endothelial cells by reducing reactive oxygen species. Am J Hypertens. 2002; 15:891-5. DOI | PubMed
33. Zhang Y, Yang X, Bian F, Wu P, Xing S, Xu G, et al. TNF-alpha promotes early atherosclerosis by increasing transcytosis of LDL across endothelial cells: crosstalk between NF-kappaB and PPAR-gamma. J Mol Cell Cardiol. 2014; 72:85-94. DOI | PubMed
34. Song D, Fang G, Mao SZ, Ye X, Liu G, Miller EJ, et al. Selective inhibition of endothelial NF-kappaB signaling attenuates chronic intermittent hypoxia-induced atherosclerosis in mice. Atherosclerosis. 2018; 270:68-75. DOI | PubMed