Effects of acute and chronic inflammation on aortic remodelling in Sprague-Dawley rats

Abstract

iii ABSTRACT Background. Cardiovascular disease (CVD) is a burgeoning health concern and remains one of the leading causes of morbidity and mortality worldwide. Inflammation has received considerable attention as a major driving factor underlying the pathogenesis of CVD. The contribution of inflammation to CVD is particularly important through its impact on arterial remodelling. During arterial remodelling, vascular cells, including those in large, elastic arteries like the aorta, undergo various structural and functional changes. However, the process and time course of systemic inflammation can be variable, and hence, the molecular mechanisms involved in inflammation-induced aortic stiffness under different conditions require elucidation. Understanding these mechanisms in humans is challenging due to limited accessibility of vascular tissue. In this regard, the use of animal models, such as the LPS rat model, provides valuable insight into the effects of inflammation on vascular pathophysiology. Therefore, the present study aimed to assess the molecular mechanisms underlying aortic functional changes in acute and chronic inflammation in Sprague-Dawley rats. Methods. Three-month-old, male, Sprague-Dawley rats were assigned to an acute 24-hour (24H, n=12), acute one-week (1W, n=18), or chronic four-week (4W, n=19) study. The rats in the 24H study were divided into a control group (n=6) which received a single intraperitoneal administration of saline (0.1 mL); and an inflammation group (n=6) which received a single intraperitoneal administration of lipopolysaccharide (LPS, 10 mg.kg-1) and were sacrificed after 24 hours. Similarly, the rats in the 1W study were divided into a control (n=10) and inflammation (n=8) group which received a single intraperitoneal administration of saline (0.1 mL) and LPS (1 mg.kg-1), respectively, and sacrificed after one week. The rats in the 4W study were divided into the control (n=10) and inflammation (n=9) group, which received saline (0.1 mL) and LPS (1 mg.kg-1), respectively, once a week for four consecutive weeks and were sacrificed seven days after the final injection. Body mass, systolic (SBP) and diastolic blood pressure (DBP) were measured prior to sacrifice. Aortic stiffness was determined by measuring maximum flow velocity (Vmax), velocity time integral (VTI), pulse wave velocity (PWV), stiffness parameter β, arterial compliance (AC), and distensibility coefficient (DC) in the abdominal aortae using ultrasound in anaesthetised rats. At termination, rats were exsanguinated by cardiac puncture and the aortae were excised. Serum concentrations of interleukin (IL)-1β and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). Relative mRNA expression of markers of inflammation iv (TNF-α and IL-6); oxidative stress (SOD-2); endothelial activation (VCAM-1); arterial remodelling (MMP-9 and VEGFA); and NF-κB pathway activation (NFKBIA and BDKRB1) was measured in aortic tissue using real-time reverse transcription polymerase chain reaction (RT-PCR). Changes in body mass and BP from baseline to termination in all groups were determined using a repeated measures analysis of variance (ANOVA). Group differences in aortic stiffness parameters and molecular markers between control and LPS groups in each study were determined using a two-way ANOVA, followed by a Tukey post hoc test. All associations were determined by Pearson’s correlations. Results. In the 24H group, body mass, SBP, DBP, and echo-tracking measures of aortic stiffness did not significantly differ between LPS-treated and -untreated rats at termination (all p>0.05). Serum concentrations of IL-1β (p=0.02) and IL-6 (p=0.03) were increased in the LPS-treated rats compared to untreated rats. The mRNA expression of TNF-α (p<0.0001), IL-6 (p<0.0001), VCAM-1 (p<0.0001), MMP-9 (p<0.0001), and NFKBIA (p=0.04) were increased in the LPS-treated rats compared to untreated rats. The expression of VCAM-1 and MMP-9 were positively associated with inflammatory gene expression (all p<0.05). In the 1W group, SBP was increased in the LPS-treated rats at termination compared to baseline (p=0.04). Echo-tracking results showed increased Vmax (p=0.04) and VTI (p=0.03) with decreased AC (p=0.04) in the LPS-treated rats compared to control rats. While serum concentrations of IL-1β (p=0.03) were increased in the LPS-treated compared to untreated rats, there were no differences in the expression of inflammatory genes, TNF-α or IL-6 (both p>0.05). The expression of MMP-9 (p=0.03) was increased in the LPS-treated compared to untreated rats. In the 4W group, termination SBP was increased in the LPS-treated rats compared to control rats (p=0.0006) and compared to baseline (p<0.0001). A significant increase was observed in PWV (p=0.03) and stiffness parameter β (p=0.02), whereas DC (p=0.01) was decreased in the LPS-treated rats compared to control rats. Serum concentrations of IL-1β (p=0.01) and IL-6 (p=0.0005) were increased in the LPS-treated rats compared to control rats. The mRNA expression of IL-6 (p=0.04) and BDKRB1 (p=0.03) were increased in the LPS-treated rats compared to control rats, without concomitant changes in the expression of arterial remodelling markers (all p>0.05). Increased mRNA expression of TNF-α was significantly associated with Vmax, VTI, PWV, and stiffness parameter β (all p<0.05). v Conclusion. This study demonstrates that the conditions of inflammation differentially affect the pathophysiological mechanisms of aortic remodelling. Genes involved in inflammation and endothelial activation are upregulated during the early stages of the acute inflammatory response but can stabilise as inflammation resolves. Despite the resolution of inflammation, genes involved in arterial remodelling remain upregulated and may impact haemodynamic and flow properties of the aorta. Chronic exposure to inflammation resulted in increased aortic stiffness and reduced contractile properties of the aorta, which may have been as a result of chronic upregulation of the NF-κB pathway. Taken together, these results suggest that inflammation independently results in pathological remodelling of the aorta, which may be initiated in the early stages of inflammation and, in the long-term, could result in substantial functional changes of the aorta.