SAVS - Downregulated Calcification Signaling, Abrogated Growth, and Preserved Aortic Compliance Can Be Achieved by Inhibiting SGK-1 in Small AAA

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Downregulated Calcification Signaling, Abrogated Growth, and Preserved Aortic Compliance Can Be Achieved by Inhibiting SGK-1 in Small AAA
Matthew Patrick Anderson, Alexander Rovner, Vinitha Uppalapati, Casey Donahoe, Brooks Winer, Ying Xiong, Rupak Mukherjee, Jeffery Jones, Jean Marie Ruddy
Medical University of South Carolina, Charleston, SC

BACKGROUND: Current screening practices have increased the number of patients diagnosed with, and monitored for, small AAA, however there are no directed medical therapies, and surgical decision-making continues to rely on aortic diameter. The benefit of slowing the AAA growth rate has been conceptually accepted but has not yet been achieved due to a knowledge gap regarding key pathophysiologic processes in the wall of small AAA. Recent literature supports phenotype switching of aortic medial vascular smooth muscle cells (VSMCs) to synthetic profiles that promote matrix remodeling, but cell signaling targets remain elusive. Furthermore, how can these VSMC transitions be assessed in the absence of obtaining tissue in small AAA? Although collection of remote data such as mechanical parameters and plasma biomarker analysis has been introduced several times, these concepts have not achieved widespread application to everyday patient care. Herein we propose that concurrently assessing mechanical and biochemical markers over the course of AAA growth will translate to cellular pathology and facilitate identifying a target for pharmacotherapeutic engineering.Stretch-induced remodeling has been extensively studied in other vascular pathologies, such as vein graft intimal hyperplasia, pulmonary hypertension, and vascular calcification, and has been attributed to the activity of the serum and glucocorticoid inducible kinase-1 (SGK-1). This kinase has been linked to the modulation of proteases and pro-inflammatory cytokines, therefore SGK-1 as a driver of degenerative aortic remodeling has been an area of active research in the Ruddy Lab. Briefly, the mechanosensitivity of SGK-1 was confirmed by applying biaxial cyclic stretch to wild-type (WT) murine aortic VSMCs where blockade with a specific SGK-1 inhibitor (EMD638683) prevented tension-induced expression of interleukin-6 (IL-6). This pro-inflammatory signal was theorized to direct aortic macrophage accumulation to promote matrix remodeling; therefore, an AngII-induced model of murine hypertension (HTN) was used to show that aortic SGK-1 activity, abundance of macrophage marker F4/80, and plasma IL-6 levels were elevated with HTN. These values returned to baseline with EMD638683 treatment despite no change in blood pressure, suggesting that mechanical stimulation remained but the intracellular signaling pathway was blocked. Building on these findings in hypertensive signaling, the Ruddy Lab has sought to further define the role of SGK-1 in the expression of markers associated with phenotype switching to macrophage-like (IL-6, Cystatin C (CysC), Cathepsin S (CtsS)), fibroblast-like (Tenascin C (TNC)), or osteoblast-like (osteoprotegerin (OPG)) VSMCs. These aortic pathologic markers (APMs) were first investigated in the AngII-induced model of HTN, wherein there was a significant increase in circulating levels of IL-6, CysC, CtsS, and TNC following 21 days of elevated blood pressure in WT mice. Interestingly, when HTN was induced in the smooth muscle cell specific SGK-1KO^+/- (SMC-SGK-1KO^+/-) mice established in the Ruddy lab, these plasma markers were unaffected despite the same level of BP elevation, suggesting that SGK-1 was a major contributor to mechanical signaling to upregulate production of subset APMs. When concurrently assessing ultrasound-derived mechanical parameters within the murine abdominal aorta, reduced distensibility (D) and increased pulse propagation velocity (PPV) were quantified, both indicating increased aortic stiffness. With HTN being a well-established risk factor for AAA development and the expectation that intrinsic aortic wall tension contributes to progression of this pathology, we sought to investigate a role for SGK-1 in AAA growth. Previous research has shown an increased expression of SGK-1 in a rabbit aneurysm model and human AAA tissue analysis has likewise suggested elevated SGK-1 activity. Employing the validated peri-adventitial CaCl₂ model of murine AAA induction, preliminary data has indicated that while WT mice achieve approximately ~67% aortic dilation (AoD) at 21 days, initiating EMD treatment at the time of AAA induction reduced growth to ~30% and that degree of growth was likewise seen in SMC-SGK-1KO^+/- mice at 21 days following AAA induction. Ultrasound-derived mechanical assessment of these animals indicated preserved PPV when SGK-1 activity was reduced. Expecting that PPV represents contractile activity of native aortic VSMCs, these findings further supported SGK-1 as a major contributor to VSMC signaling in pathologic aortic remodeling. We therefore have developed the hypothesis that introducing SGK-1 inhibition in small AAA will abrogate growth, alter production of APMs, and preserve aortic compliance. METHODS: WT and SMC-SGK-1 KO^+/-mice underwent peri-adventitial CaCl₂ application for AAA induction at day 0. Sham groups had aortic exposure and treatment with NaCl. A randomly selected subset of mice then had osmotic pump placement with EMD638683 (EMD, 2.5mg/kg/day) beginning at day 21. Mouse groups included WT+Sham, WT+21AAA, WT+42AAA, WT+42AAA+EMD, SMC-SGK-1KO^+/-+Sham, SMC-SGK-1KO^+/-+21AAA, SMC-SGK-1KO^+/-+42AAA (n=4-6 for each). Vevo3100 micro ultrasound system was utilized to capture axial and longitudinal images of the abdominal aorta while the mouse was maintained under light isoflurane anesthetic to maintain a heart rate of 450-550. These images were analyzed in VevoVasc software system to calculate radial strain (RS), distensibility (D), and pulse propagation velocity (PPV). Ultrasound was conducted on days 0, 21, and 42. Aortic diameter (AoD) was derived from digital microscopy captured at day 0 versus terminal (day 21 or day 42) such that the percent change from baseline calculation was individualized for each mouse. At the time of terminal procedure, in addition to aortic imaging and harvest, direct left ventricular puncture allowed for plasma collection. ELISA was used to quantify APMs (IL-6, CysC, CtsS, TNC, OPG). Statistical analysis was conducted by ANOVA utilizing GraphPad Prism (version 10.1.2).RESULTS: Consistent with the preliminary data, WT+21AAA mice in this experimentation had 72+5% AoD at 21 days. When extended to 42 days of AAA growth, WT+42AAA mice had 116+2% increase in AoD (p<0.05 vs WT+21AAA), confirming ongoing growth of small AAA with this model. Pharmacologic inhibition of SGK-1 introduced at day 21 reduced further growth (55+2% in WT+42AAA+EMD) and this represents a significant reduction compared to WT+42AAA (p<0.05). Similarly, SMC-SGK-1KO^+/-+42AAA mice had abrogated AAA growth at 42 days (58+6%; p<0.05 vs WT+42AAA). Among plasma APMs, only OPG was significantly increased in WT+42AAA (p<0.05 vs WT+Sham) and then reduced with SGK-1 inhibition (p<0.05 vs WT+42AAA+EMD and vs SMC-SGK-1KO^+/-+42AAA), implicating SGK-1 signaling in pro-calcifying pathways. CysC was significantly elevated in WT+42AAA and WT+42AAA+EMD (p<0.05 vs WT+Sham) while in the SMC-SGK-1KO^+/-+42AAA group CysC only showed an upward trend, which may suggest that the timing of SGK-1 activity is pivotal to production of this serine protease inhibitor in AAA. CtsS, a notable serine protease produced by intracellular lysosomes, was unchanged in plasma of WT+42AAA, SMC-SGK-1KO^+/-+42AAA and WT+42AAA+EMD groups as compared to the WT+Sham. CtsS abundance maintained within the aortic wall has not yet been assessed. TNC also had an upward trend in WT+42AAA which was reversed in both the SMC-SGK-1KO^+/-+42AAA and WT+42AAA+EMD, suggesting SGK-1 dependency but did not reach significance over the present timeline. By day 21, RS and D showed progressive stiffness in the WT+21AAA, SMC-SGK-1KO^+/-+21AAA and WT+21AAA+EMD treatment groups, with WT+21AAA and SMC-SGK-1KO^+/-+21AAA achieving statistical significance when compared to day 0 (p<0.05 vs baseline). From day 21 to 42, there was no further increase in RS or D in the WT+42AAA, SMC-SGK-1KO^+/-+42AAA and WT+42AAA+EMD treatment groups. PPV was significantly elevated in the WT+21AAA, SMC-SGK-1KO^+/-+21AAA and WT+21AAA+EMD treatment groups (p<0.05 vs baseline), but for the groups with reduction of SGK-1 activity PPV had a notable downward trend to suggest regained compliance. Similarly, PPV at day 42 remained elevated in the WT+42AAA group (p<0.05 vs baseline), but PPV was significantly reduced in the SMC-SGK-1KO^+/-+42AAA and WT+42AAA+EMD groups when compared to WT+42AAA (p<0.05 for each). Importantly, the elevated PPV in WT+42AAA+EMD mice measured at day 21 ultrasound was then reversed by day 42 with SGK-1 inhibition via EMD.CONCLUSIONS: Given the increasing number of patients being diagnosed with small AAA as a result of increased screening, better understanding of the biomechanical signaling pathway that drives aneurysm growth would allow for tracking of aneurysm changes and provide the potential to predict those at risk for continued expansion and thus surgical intervention. Identifying these crucial pathways may also enable pharmacologic targeting. Our study has demonstrated that plasma APMs and aortic stiffness parameters can be tracked in small AAA with trends expected to convey VSMC pathology. More specifically, reducing SGK-1 activity shifted VSMC signaling away from a pro-calcifying pathway with resultant reduction in aortic dilation and preservation of aortic compliance. Temporospatial analysis and correspondence to the human condition are ongoing, but the current murine translational findings suggest that SGK-1 warrants further investigation as a druggable target to treat small AAA.

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