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Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.

ABSTRACT: Arteriovenous access dysfunction is a major cause of morbidity for hemodialysis patients. The pathophysiology of arteriovenous fistula (AVF) maturation failure is associated with inflammation, impaired outward remodeling (OR) and intimal hyperplasia. RP105 is a critical physiologic regulator of TLR4 signaling in numerous cell types. In the present study, we investigated the impact of RP105 on AVF maturation, and defined cell-specific effects of RP105 on macrophages and vascular smooth muscle cells (VSMCs). Overall, RP105-/- mice displayed a 26% decrease in venous OR. The inflammatory response in RP105-/- mice was characterized by accumulation of anti-inflammatory macrophages, a 76% decrease in pro- inflammatory macrophages, a 70% reduction in T-cells and a 50% decrease in MMP-activity. In vitro, anti-inflammatory macrophages from RP105-/- mice displayed increased IL10 production, while MCP1 and IL6 levels secreted by pro-inflammatory macrophages were elevated. VSMC content in RP105-/- AVFs was markedly decreased. In vitro, RP105-/- venous VSMCs proliferation was 50% lower, whereas arterial VSMCs displayed a 50% decrease in migration, relative to WT. In conclusion, the impaired venous OR in RP105-/- mice could result from of a shift in both macrophages and VSMCs towards a regenerative phenotype, identifying a novel relationship between inflammation and VSMC function in AVF maturation.

SUBMITTER: Bezhaeva T

PROVIDER: S-EPMC5578984 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.

Bezhaeva Taisiya T Wong ChunYu C de Vries Margreet R MR van der Veer Eric P EP van Alem Carla M A CMA Que Ivo I Lalai Reshma A RA van Zonneveld Anton-Jan AJ Rotmans Joris I JI Quax Paul H A PHA

Scientific reports 20170831 1

Arteriovenous access dysfunction is a major cause of morbidity for hemodialysis patients. The pathophysiology of arteriovenous fistula (AVF) maturation failure is associated with inflammation, impaired outward remodeling (OR) and intimal hyperplasia. RP105 is a critical physiologic regulator of TLR4 signaling in numerous cell types. In the present study, we investigated the impact of RP105 on AVF maturation, and defined cell-specific effects of RP105 on macrophages and vascular smooth muscle cel ...[more]

PMID: 28860634

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Project description:Following creation, an arteriovenous fistula (AVF) must mature (i.e., enlarge lumen to allow high blood flow) before being used for hemodialysis. AVF maturation failure rates are high, and currently, there are no effective therapy to treat this problem. The maturation process is likely affected by the integrity of the vascular extracellular matrix (ECM). Natural Vascular Scaffolding (NVS) Therapy is a new technology that interlinks collagen and elastin via photoactivation of a locally delivered small molecule (4-amino-1,8-naphtalamide). We hypothesized that NVS Therapy may improve AVF remodeling by preserving ECM integrity. AVFs were created in Wistar male rats by connecting the femoral vein (end) to femoral artery (side) in the same limb. Immediately after blood flow was restored to dilate the femoral vein by arterial pressure, a 10 ?l-drop of the NVS compound (2 mg/ml) was placed on the anastomosis perivascularly. Following 5-min incubation, the NVS treated area was exposed to 1-min illumination by 450-nm light. The control group received 10 ?l-drop of phosphate buffered saline (PBS) and the same light activation. The skin was closed, and rats were euthanized 4 weeks (n = 6-9 per group) post-AVF creation for histology, morphometry, immunohistochemistry (IHC), and multiphoton microscopy for second-harmonic-generation evaluation of collagen fibers. The vascular thickness was similar in both groups. The AVF vein's open lumen area and % open lumen area in NVS-treated rats were significantly larger than in PBS-treated rats (4.2-fold p = 0.014 and 2-fold p = 0.009, respectively). The inflammatory markers IL-6 and MMP-9 in the AVF walls were significantly decreased in the NVS group than the PBS group. Collagen fibers in the vascular wall trended toward perpendicular alignment to the lumen circumference in the NVS-treated AVFs, with more defined shape but less area than in the PBS-treated AVFs. These results indicate that the NVS Therapy exerted changes in collagen, which may influence AVF maturation. Rats tolerated the NVS treatment well, and the lack of cell death by the treatment was confirmed in cell culture experiments. These results suggest that NVS treatment is safe and may have therapeutic potential by facilitating lumen expansion to enhanced AVF maturation in patients.

Project description:Objective:The arteriovenous fistula (AVF) is the preferred method of dialysis access because of its proven superior long-term outcomes.However, women havelower rates of AVF patency andutilizationthan men.We used a novel mouseAVF model that recapitulates human AVF maturation to determine whether there are differences in AVF patency in female and male mice. Methods:Aortocaval fistulas were created in female and male C57BL/6 mice (9-10 weeks). At days 0, 3, 7, and 21, infrarenal inferior vena cava (IVC) and aortic diameters and flow velocity were monitored by Doppler ultrasound and used to calculate the vessel diameter, blood flow, and shear stress. AVF were harvested, and expression of proteins was examined by proteomic analysis and immunofluorescence and of messenger RNA by quantitative polymerase chain reaction analysis. Results:At baseline, female mice weighed less and had lower IVC velocity and smaller magnitudes of shear stress, but there was no significant difference in IVC diameter and thickness. After AVF creation, both female and male mice had similar IVC dilation and thickening with no significant differences in IVC wall thickness at day 21. However, female mice had diminished AVF patency by day 42 (25.7% vs 64.3%; P = .039). During fistula remodeling, female mice had lower IVC mean velocity and shear stress magnitude and increased spectral broadening (days 0-21). Messenger RNA and protein expression of Krüppel-like factor 2, endothelial nitric oxide synthase, and vascular cell adhesion molecule 1 was similar at baseline in female and male mice but increased in the AVF only in male mice but not in female mice (day 21). Proteomic analysis of female and male mice detected 56 proteins expressed at significantly higher levels in the IVC of female mice and 67 proteins expressed at significantly higher levels in the IVC of male mice (day 7); function-specific analysis showed that the IVC of male mice overexpressed proteins that belong to pathways implicated in the regulation of vascular function, thrombosis, response to flow, and vascular remodeling. Conclusions:AVF in female mice have diminished patency, preceded by lower velocity, reduced magnitudes of shear stress, and less laminar flow during remodeling. There is also sex-specific differential expression of proteins involved in thrombosis, response to laminar flow, inflammation, and proliferation. These findings suggest that hemodynamic changes during fistula maturation may play an important role underlying the diminished rates of AVF utilization in women. Clinical Relevance:Women have lower rates of arteriovenous fistula (AVF) utilization than men. Using a mouse AVF model that recapitulates human AVF maturation, we show that female mice have similar AVF remodeling but diminished patency. AVF remodeling in female mice is associated with reduced shear stress and laminar flow; lack of increased transcription and translation of several anti-inflammatory, antiproliferative, and laminar flow response proteins (endothelial nitric oxide synthase, Krüppel-like factor 2, and vascular cell adhesion molecule 1); and different patterns of expression of pathways that regulate thrombosis and venous remodeling. Identifying downstream targets involved in these mechanisms may improve AVF outcomes in female patients.

Project description:Objective---Arteriovenous fistulae (AVF) placed for hemodialysis have high flow rates that can stimulate left ventricular (LV) hypertrophy. LV hypertrophy generally portends poor cardiac outcomes, yet clinical studies point to superior cardiac-specific outcomes for patients with AVF when compared to other dialysis modalities. We hypothesize that AVF induce physiologic cardiac hypertrophy with cardioprotective features.Methods---9-11 week C57Bl/6 male and female mice were treated with sham laparotomy or an aortocaval fistula via a 25Ga needle. Cardiac chamber size and function were assessed with serial echocardiography, and cardiac CTA. Hearts were harvested at 5 weeks post-operatively, and collagen content assessed with Masson's trichrome. Bulk mRNA sequencing was performed from LV of sham and AVF mice at 10 days. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (Qiagen) to identify affected pathways and predict downstream biological effects.Results---Mice with AVF had similar body weight and wet lung mass, but increased cardiac mass compared to sham-operated mice. AVF increased cardiac output while preserving LV systolic and diastolic function, as well as indices of right heart function; all 4 cardiac chambers were enlarged, with slight decrement in relative LV wall thickness. Histology showed preserved collagen density within each of the 4 chambers without areas of fibrosis. RNA sequencing captured 19,384 genes, of which 857 were significantly differentially expressed, including transcripts from extracellular matrix-related genes, ion channels, metabolism, and cardiac fetal genes. Top upstream regulatory molecules predicted include activation of angiogenic (Vegf, Akt1), pro-cardiomyocyte survival (Hgf, Foxm1, Erbb2, Lin9, Areg), and inflammation-related (CSF2, Tgfb1, TNF, Ifng, Ccr2, IL6) genes, as well as the inactivation of cardiomyocyte antiproliferative factors (Cdkn1a, FoxO3, α-catenin). Predicted downstream effects include reduction to heart damage, and increased arrhythmia, angiogenesis, and cardiogenesis. There were no significant sex-dependent differences in the AVF-stimulated cardiac adaptation.Conclusions---AVF stimulate adaptive cardiac hypertrophy in wild-type mice without heart failure or pathological fibrosis. Transcriptional correlates suggest AVF-induced cardiac remodeling has some cardioprotective, although also arrhythmogenic features.

Project description:Cardiovascular events are the primary cause of death among dialysis patients. While arteriovenous fistulas (AVFs) are the access of choice for hemodialysis patients, AVF creation can lead to a volume overload (VO) state in the heart. We developed a three-dimensional (3D) cardiac tissue chip (CTC) with tunable pressure and stretch to model the acute hemodynamic changes associated with AVF creation to complement our murine AVF model of VO. In this study, we aimed to replicate the hemodynamics of murine AVF models in vitro and hypothesized that if 3D cardiac tissue constructs were subjected to "volume overload" conditions, they would display fibrosis and key gene expression changes seen in AVF mice. Mice underwent either an AVF or sham procedure and were sacrificed at 28 days. Cardiac tissue constructs composed of h9c2 rat cardiac myoblasts and normal adult human dermal fibroblasts in hydrogel were seeded into devices and exposed to 100 mg/10 mmHg pressure (0.4 s/0.6 s) at 1 Hz for 96 h. Controls were exposed to "normal" stretch and experimental group exposed to "volume overload". RT-PCR and histology were performed on the tissue constructs and mice left ventricles (LVs), and transcriptomics of mice LVs were also performed. Our tissue constructs and mice LV both demonstrated cardiac fibrosis as compared to control tissue constructs and sham-operated mice, respectively. Gene expression studies in our tissue constructs and mice LV demonstrated increased expression of genes associated with extracellular matrix production, oxidative stress, inflammation, and fibrosis in the VO conditions vs. control conditions. Our transcriptomics studies demonstrated activated upstream regulators related to fibrosis, inflammation, and oxidative stress such as collagen type 1 complex, TGFB1, CCR2, and VEGFA and inactivated regulators related to mitochondrial biogenesis in LV from mice AVF. In summary, our CTC model yields similar fibrosis-related histology and gene expression profiles as our murine AVF model. Thus, the CTC could potentially play a critical role in understanding cardiac pathobiology of VO states similar to what is present after AVF creation and may prove useful in evaluating therapies.

Dataset Information

Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.

Publications

Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.

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