Fibronectin‐Mediated Inflammatory Signaling Through Integrin α5 in Vascular Remodeling

Background Adhesion of vascular endothelial cells to the underlying basement membrane potently modulates endothelial cells to cells' inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin α2β1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes endothelial cells to inflammatory stimuli through integrins α5β1and and αvβ3. A chimeric integrin in which the cytoplasmic domain of α5 is replaced with that of α2 pairs with β1 and binds fibronectin but signals like α2β1. Methods and Results Here, we examined mice in which integrin α5 is replaced with the α5/2 chimera, using the transverse aortic constriction and partial carotid ligation models of vessel remodeling. Following transverse aortic constriction and partial carotid ligation surgery, wild‐type mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. α5/2 mice also showed reduced artery wall hypertrophy in the transverse aortic constriction model and diminished inward remodeling in the partial carotid ligation model. Acute atherosclerosis after partial carotid ligation in hyperlipidemic ApoE −/− mice on a high fat diet was dramatically decreased in α5/2 mice. Conclusions Fibronectin and integrin α5 signaling is a key element of pathological vascular remodeling in acute models of both hypertension and disturbed flow. These results underscore the key role for integrin α5 signaling in pathological vascular remodeling associated with hypertension and atherosclerosis and support its potential as a therapeutic target.

Identification of intima-to-media signals for flow-induced vascular remodeling using correlative gene expression analysis

Changes in blood flow can induce arterial remodeling. Intimal cells sense flow and send signals to the media to initiate remodeling. However, the nature of such intima-media signaling is not fully understood. To identify potential signals, New Zealand white rabbits underwent bilateral carotid ligation to increase flow in the basilar artery or sham surgery (n = 2 ligated, n = 2 sham). Flow was measured by transcranial Doppler ultrasonography, vessel geometry was determined by 3D angiography, and hemodynamics were quantified by computational fluid dynamics. 24 h post-surgery, the basilar artery and terminus were embedded for sectioning. Intima and media were separately microdissected from the sections, and whole transcriptomes were obtained by RNA-seq. Correlation analysis of expression across all possible intima-media gene pairs revealed potential remodeling signals. Carotid ligation increased flow in the basilar artery and terminus and caused differential expression of 194 intimal genes and 529 medial genes. 29,777 intima-media gene pairs exhibited correlated expression. 18 intimal genes had > 200 medial correlates and coded for extracellular products. Gene ontology of the medial correlates showed enrichment of organonitrogen metabolism, leukocyte activation/immune response, and secretion/exocytosis processes. This demonstrates correlative expression analysis of intimal and medial genes can reveal novel signals that may regulate flow-induced arterial remodeling.

Abstract 17221: Stable Flow-Induced Expression of KLK10 Inhibits Endothelial Inflammation and Atherosclerosis

Introduction: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow ( d-flow ) while the straight regions exposed to stable flow ( s-flow ) are protected. The proatherogenic and atheroprotective effects of flow are mediated in large part by the global changes in endothelial cell gene expression, which regulate endothelial dysfunction and inflammation. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10) as one of the most flow-sensitive genes in arterial endothelial cells using the partial carotid ligation model of d-flow -induced atherosclerosis. KLK10 is a secreted serine protease, but its role in endothelial function and atherosclerosis is unknown. Methods/Results: Here, we validated that KLK10 was upregulated under s-flow conditions and downregulated under d-flow conditions using the in vivo mouse models and in vitro studies using endothelial cells (ECs). Through in vitro functional studies using ECs, we found that KLK10 produced by s-flow protected against endothelial inflammation and permeability dysfunction. Furthermore, treatment with rKLK10 or overexpression of KLK10 plasmids in vivo decreased endothelial inflammation the mouse model. Further, rKLK10 injection or ultrasound-mediated transfection of KLK10 plasmids in the hind leg muscles led to inhibition of atherosclerosis in ApoE-/- mice with the partial carotid ligation surgery. Studies using the pharmacological inhibitors and siRNAs showed that the anti-inflammatory effects of KLK10 was mediated by the Protease Activated Receptors 1 and 2, but without directly cleaving them. Further studies show that KLK10’s anti-inflammatory effect was mediated by the NFκB and VCAM1 and ICAM1 expression pathway. In addition, immunostaining showed that KLK10 expression is significantly reduced in human coronary arterial sections with atherosclerotic plaques compared to the non-diseased controls. Conclusions: We found that KLK10 is a potent flow-sensitive secreted protein, which serve as a novel anti-inflammatory and anti-atherogenic factor. KLK10 may be a potential anti-atherogenic therapeutic.

1439PRAS40 attenuates mTOR-dependent endothelial inflammation and atherosclerosis

Introduction Endothelial inflammation plays a pivotal role in atherosclerosis. Many inflammatory and metabolic signals converge upon mechanistic target of rapamycin (mTOR), and inhibition of mTOR has been shown to reduce atherosclerosis. However, clinical use of mTOR-inhibitors is limited by serious adverse effects, of which insulin resistance and dyslipidemia are particularly troubling in the context of atherosclerosis. In that respect, targeting PRAS40, an endogenous modulator of mTOR complex 1 (mTORC1) with highly cell type-specific effects on mTOR signaling, may be a more promising approach. In fact, we have previously demonstrated that, in contrast to conventional mTOR inhibitors, PRAS40 gene therapy substantially improves metabolic profile in obese mice. However, the function of PRAS40 in endothelial cells and its role in atherosclerosis have never been investigated. Methods and results To define the impact of PRAS40 on endothelial mTORC1-signaling in this context, cultured human umbilical vein endothelial cells (HUVECs) were exposed to the atherogenic cytokine TNFα. TNFα induced mTOR signaling as evidenced by increased phosphorylation of S6 kinase and ribosomal S6 protein. Interestingly, this effect was strongly augmented upon siRNA-mediated knock-down of PRAS40, indicating a negative regulation of mTORC1 by PRAS40 in endothelial cells. Moreover, PRAS40-knockdown promoted TNFα-induced inflammatory signaling as reflected by increased proliferative activity, upregulation of atherogenic markers like CCL2 and VCAM-1, as well as enhanced monocyte recruitment in the THP-1 adhesion assay. In contrast, PRAS40-overexpression blocked TNFα-induced activation of mTORC1 and consistently suppressed all of these measures of inflammatory activation. All effects of PRAS40-overexpression could be reproduced by the mTORC1 inhibitors rapamycin and torin1. Thus, our in vitro studies suggest that in endothelial cells PRAS40 exerts anti-atherogenic effects by negative regulation of mTORC1. To validate these findings in vivo in the context of atherosclerosis we created transgenic mice with tamoxifen-inducible endothelium-specific PRAS40-deficiency (EC-PRAS40-KO). These mice were exposed to a model of accelerated atherosclerosis based on western diet and partial carotid ligation: Four weeks after partial carotid ligation, neointimal and atherosclerotic lesion formation was strongly enhanced in EC-PRAS40-KO mice. Moreover, mTORC1 activity as well as CCL2 and VCAM-1 expression were markedly increased compared to control mice. Conclusion Our data indicate that PRAS40 suppresses atherosclerosis via inhibition of mTORC1-mediated inflammatory signaling in endothelial cells. In conjunction with its favourable effects on metabolic homeostasis, the overall therapeutic profile of PRAS40-treatment appears to be beneficial compared to conventional mTOR-inhibitors. Taken together PRAS40 may qualify as a promising therapeutic target for the treatment of atherosclerosis. Acknowledgement/Funding German Federal Ministry of Education and Research, DZHK (German Centre for Cardiovascular Research)

3312Thrombomodulin expressed on vascular smooth muscle cell influences arterial injury-induced neointima formation in mouse

Background Thrombomodulin (TM) is a cell membrane-bound anticoagulant protein that only expresses on endothelial cells in normal artery. Vascular smooth muscle cells (SMCs) start to exhibit TM after arterial injury. Our previous study demonstrated that vascular SMC-bound TM expression was associated with SMC synthetic phenotype. TM knockdown not only attenuated aortic SMCs proliferation but also reduced aortic SMC-mediated inflammation. In this study, we investigated the effect of vascular SMC-bound TM on arterial injury-induced neointima formation in mouse. Methods and results Because complete loss of TM in TM knockout transgenic mice causes embryonic lethality, we generated vascular SMC-specific TM-deficient mice (SM22-cretg/TMflox/flox) and their wild-type controls (SM22-cretg/TM+/+) using the Cre-loxP system to explore the role of vascular SMC membrane-bound TM in vivo. The blood pressure and body weight were similar between SM22-cretg/TMflox/flox mice and their wild-type controls. Carotid ligation caused neointima formation in mice. Immunofluorescence staining showed that there was large amount TM expression in the medial and neointimal cells at 4 weeks in SM22-cretg/TM+/+ mice after carotid ligation, but there was no TM staining could be found in SM22-cretg/TMflox/flox mice. There was a progressively increased neointima area from 2 to 4 weeks after carotid ligation both in SM22-cretg/TMflox/flox mice and SM22-cretg/TM+/+ mice, but the neointima area and neointima/media area ratio were significantly smaller in SM22-cretg/TMflox/flox mice than SM22-cretg/TM+/+ mice. Immunofluorescence staining showed that there were less Ki67-positive cells in the media and neointima in SM22-cretg/TMflox/flox mice indicating less proliferating cells in the arterial wall of TM-deficient mice. The α-smooth muscle actin-positive staining area was also larger in the SM22-cretg/TMflox/flox mice, suggesting TM deficiency of SMCs in medial lesion exhibited a more contractile status after carotid ligation. Conclusions Our results indicated that vascular SMC-bound TM not only mediated vascular SMC phenotype change and cell behavior but also significantly influenced arterial injury-induced neointima formation. Acknowledgement/Funding This study was sponsored by grants 104-2314-B-006-083-MY2 and 106-2314-B-006-045-MY3 from the Ministry of Science and Technology, Taipei, Taiwan.

Strain-Related Differences in Mouse Neonatal Hypoxia-Ischemia

Neonatal hypoxic-ischemic brain injury is commonly studied by means of the Vannucci procedure in mice or rats (unilateral common carotid artery occlusion followed by hypoxia). Previously, we modified the postnatal day 7 (P7) rat procedure for use in mice, and later demonstrated that genetic strain strongly influences the degree of brain injury in the P7 mouse model of hypoxia-ischemia (HI). Recently, the P9 or P10 mouse brain was recognized as the developmental equivalent of a term neonatal human brain, rather than P7. Consequently, the Vannucci procedure has again been modified, and a commonly used protocol employs 10% oxygen for 50 min in C57Bl/6 mice. Strain differences have yet to be described for the P9/P10 mouse model. In order to determine if the strain differences we previously reported in the P7 mouse model are present in the P9 model, we compared 2 commonly used strains, CD1 and C57Bl/6J, in both the P7 (carotid ligation [in this case, right] followed by exposure to 8% oxygen for 30 min) and P9 (carotid ligation [in this case left] followed by exposure to 10% oxygen) models of HI. Experiments using the P7 model were performed in 2001–2012 and those using the P9 model were performed in 2012–2016. Five to seven days after the HI procedure, mice were perfused with 4% paraformaldehyde, their brains were sectioned on a Vibratome (50 µm) and alternate sections were stained with Perl’s iron stain or cresyl violet. Brain sections were examined microscopically and scored for the degree of injury. Since brains in the P7 group had been scored previously with a slightly different system, they were reanalyzed using our current scoring system which scores injury in 11 regions: the anterior, middle, and posterior cortex; the anterior, middle, and posterior striatum; CA1, CA2, CA3, and the dentate gyrus of the hippocampus and thalamus, on a scale from 0 (none) to 3 (cystic infarct) for a total score of 0–33. Brains in the P9 group were scored with the same system. Given the same insult, the P7 CD1 mice had greater injury than the C57Bl/6J mice, which agrees with our previous findings. The P9 CD1 mice also had greater injury than the C57Bl/6J mice. This study confirms that CD1 mice are more susceptible to injury than C57Bl/6J mice and that strain selection is important when using mouse models of HI.