Dual Function for Mature Vascular Smooth Muscle Cells During Arteriovenous Fistula Remodeling.
ABSTRACT: The arteriovenous fistula (AVF) is the preferred form of hemodialysis access for patients with chronic kidney disease. However, AVFs are associated with significant problems including high incidence of both early and late failures, usually attributed to inadequate venous arterialization and neointimal hyperplasia, respectively. Understanding the cellular basis of venous remodeling in the setting of AVF could provide targets for improving AVF patency rates.A novel vascular smooth muscle cell (VSMC) lineage tracing reporter mouse, Myh11-Cre/ERT2-mTmG, was used to track mature VSMCs in a clinically relevant AVF mouse model created by a jugular vein branch end to carotid artery side anastomosis. Prior to AVF surgery, differentiated medial layer VSMCs were labeled with membrane green fluorescent protein (GFP) following tamoxifen induction. Four weeks after AVF surgery, we observed medial VSMC layer thickening in the middle region of the arterialized vein branch. This thickened medial VSMC layer was solely composed of differentiated VSMCs that were GFP+/MYH11+/Ki67-. Extensive neointimal hyperplasia occurred in the AVF region proximal to the anastomosis site. Dedifferentiated VSMCs (GFP+/MYH11-) were a major cellular component of the neointima. Examination of failed human AVF samples revealed that the processes of VSMC phenotypic modulation and intimal hyperplasia, as well as medial VSMC layer thickening, also occurred in human AVFs.We demonstrated a dual function for mature VSMCs in AVF remodeling, with differentiated VSMCs contributing to medial wall thickening towards venous maturation and dedifferentiated VSMCs contributing to neointimal hyperplasia. These results provide valuable insights into the mechanisms underlying venous adaptations during AVF remodeling.
Project description:Objective- Dysregulated proliferation of vascular smooth muscle cells (VSMC) plays an essential role in neointimal hyperplasia. CD36 functions critically in atherogenesis and thrombosis. We hypothesize that CD36 regulates VSMC proliferation and contributes to the development of obstructive vascular diseases. Approach and Results- We found by immunofluorescent staining that CD36 was highly expressed in human vessels with obstructive diseases. Using guidewire-induced carotid artery injury and shear stress-induced intima thickening models, we compared neointimal hyperplasia in Apoe-/-, Cd36-/- /Apoe-/-, and CD36 specifically deleted in VSMC (VSMC cd36-/-) mice. CD36 deficiency, either global or VSMC-specific, dramatically reduced injury-induced neointimal thickening. Correspondingly, carotid artery blood flow was significantly increased in Cd36-/- /Apoe-/- compared with Apoe-/- mice. In cultured VSMCs from thoracic aorta of wild-type and Cd36-/- mice, we found that loss of CD36 significantly decreased serum-stimulated proliferation and increased cell populations in S phase, suggesting that CD36 is necessary for VSMC S/G2-M-phase transition. Treatment of VSMCs with a TSR (thrombospondin type 1 repeat) peptide significantly increased wild-type, but not Cd36-/- VSMC proliferation. TSR or serum treatment significantly increased cyclin A expression in wild-type, but not in Cd36-/- VSMCs. STAT3 (signal transducer and activator of transcription), which reportedly enhances both VSMC differentiation and maturation, was higher in Cd36-/- VSMCs. CD36 deficiency significantly decreased expression of Col1A1 (type 1 collagen A1 chain) and TGF-?1 (transforming growth factor beta 1), and increased expression of contractile proteins, including calponin 1 and smooth muscle ? actin, and dramatically increased cell contraction. Conclusions- CD36 promotes VSMC proliferation via upregulation of cyclin A expression that contributes to the development of neointimal hyperplasia, collagen deposition, and obstructive vascular diseases.
Project description:Abnormal increases in vascular smooth muscle cells (VSMCs) in the intimal region after a vascular injury is a key event in developing neointimal hyperplasia. To maintain vascular function, proliferation and apoptosis of VSMCs is tightly controlled during vascular remodeling. NF-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) system, a key component of the oxidative stress response that acts in maintaining homeostasis, plays an important role in neointimal hyperplasia after a vascular injury; however, the role of Nrf2/Keap1 in VSMC apoptosis has not been clarified. Here we report that 14 days after arterial injury in mice, TUNEL-positive VSMCs are detected in both the neointimal and medial layers. These layers contain cells expressing high levels of Nrf2 but low Keap1 expression. In VSMCs, Keap1 depletion induces features of apoptosis, such as positive TUNEL staining and annexin V binding. These changes are associated with an increased expression of nuclear Nrf2. Simultaneous Nrf2 depletion inhibits Keap1 depletion-induced apoptosis. At 14 days after the vascular injury, Nrf2-deficient mice demonstrated fewer TUNEL-positive cells and increased neointimal formation in the neointimal and medial areas. The results suggest that the Nrf2/Keap1 system regulates VSMC apoptosis during neointimal formation, thereby inhibiting neointimal hyperplasia after a vascular injury.
Project description:Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and ?-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18-20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4<sup>+/-</sup> mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.
Project description: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.
Project description:OBJECTIVE: Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCF(Skp2) ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo. METHODS AND RESULTS: Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27(Kip1) levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27(Kip1) protein levels, enhanced medial VSMC proliferation, and the neointimal thickening. CONCLUSION: This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.
Project description:Neointimal hyperplasia, stimulated by injury and certain vascular diseases, promotes artery obstruction and tissue ischemia. In vascular smooth muscle cell (VSMCs), multiple modulators of protein handling machinery regulate intimal hyperplasia. These include elements of the VSMC unfolded protein response to endoplasmic reticulum stress (UPRER), and transglutaminase 2 (TG2), which catalyzes post-translational protein modification. Previous results for deficiency of UPRER-specific mediator XBP1, and of TG2, have been significant, but in multiple instances contradictory, for effects on cultured VSMC function, and, using multiple models, for neointimal hyperplasia in vivo. Here, we engineered VSMC-specific deficiency of XBP1, and studied cultured VSMCs, and neointimal hyperplasia in response to carotid artery ligation in vivo. Intimal area almost doubled in Xbp1fl/fl SM22?-CRE+ mice 21 days post-ligation. Cultured murine Xbp1 deficient VSMCs migrated more in response to platelet derived growth factor (PDGF) than control VSMCs, and had an increased level of inositol-requiring enzyme 1? (Ire1?), a PDGF receptor-binding UPRER transmembrane endonuclease whose substrates include XBP1. Cultured XBP1-deficient VSMCs demonstrated decreased levels of TG2 protein, in association with increased TG2 polyubiquitination, but with increased TG transamidation catalytic activity. Moreover, IRE1?, and TG2-specific transamidation cross-links were increased in carotid artery neointima in Xbp1fl/fl SM22?-CRE+ mice. Cultured TG2-deficient VSMCs had decreased XBP1 associated with increased IRE1?, and increased migration in response to PDGF. Neointimal hyperplasia also was significantly increased in Tgm2fl/fl SM22?-CRE+ mice at 21 days after carotid ligation. In conclusion, a VSMC regulatory circuit between XBP1 and TG2 limits neointimal hyperplasia in response to carotid ligation.
Project description:Microtubule stabilizing agents (MTSA) are known to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and effectively reduce neointimal hyperplasia and restenosis. Epothilones (EPOs), non-taxane MTSA, have been found to be effective in the inhibition of VSMC proliferation and neointimal formation by cell cycle arrest. However, effect of EPOs on apoptosis in hyper-proliferated VSMCs as a possible way to reduce neointimal formation and its action mechanism related to VSMC viability has not been suited yet. Thus, the purposes of the present study was to investigate whether EPOs are able to inhibit neointimal formation by inducing apoptosis within the region of neointimal hyperplasia in balloon-injured rat carotid artery, as well as underlying action mechanism. Treatment of EPO-B and EPO-D significantly induced apoptotic cell death and mitotic catastrophe in hyper-proliferated VSMCs, resulting in cell growth inhibition. Further, EPOs significantly suppressed VSMC proliferation and induced apoptosis by activation of p53-dependent apoptotic signaling pathway, Bax/cytochrome c/caspase-3. We further demonstrated that the local treatment of carotid arteries with EPOs potently inhibited neointimal lesion formation by induction of apoptosis in rat carotid injury model. Our findings demonstrate a potent anti-neointimal hyperplasia property of EPOs by inducing p53-depedent apoptosis in hyper-proliferated VSMCs.
Project description:Pro-inflammatory response of VSMCs is triggered by endothelial damage and a causative step for thrombosis and neointimal thickening in the arterial vessels. Therefore, we investigate a role of cytosolic Hsp60 as a novel pro-inflammatory mediator in VSMCs. Hsp60 was detected in the cytosol of VSMCs. The selective depletion of cytosolic Hsp60 in VSMCs reduced the IKK activation, repressed the induction of NF-κB-dependent pro-survival genes (MnSOD and Bfl-1/A1), and enhanced apoptotic death in response to TNF-α. Moreover, a quantitative RNA sequencing revealed that the expression of 75 genes among the 774 TNF-α-inducible genes was significantly reduced by the depletion of cytosolic Hsp60. In particular, the expression of pro-inflammatory cytokines/chemokines, such as CCL2, CCL20, and IL-6, was regulated by the cytosolic Hsp60 in VSMCs. Finally, the depletion of cytosolic Hsp60 markedly inhibited the neointimal thickening in the balloon-injured arterial vessels by inducing apoptotic cell death and inhibiting chemokine production. This study provides the first evidence that cytosolic Hsp60 could be a therapeutic target for preventing inflammation-driven VSMC hyperplasia in the injured vessels. Hsp60 normal vs knockout with TNF-alpha treatment
Project description:Background Migration of vascular smooth muscle cells (VSMCs) is the main contributor to neointimal formation. The Arp2/3 (actin-related proteins 2 and 3) complex activates actin polymerization and is involved in lamellipodia formation during VSMC migration. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein expressed in VSMCs. We hypothesized that MFG-E8 regulates VSMC migration through modulation of Arp2/3-mediated actin polymerization. Methods and Results To determine whether MFG-E8 is essential for VSMC migration, a model of neointimal hyperplasia was induced in the common carotid artery of wild-type and MFG-E8 knockout mice, and the extent of neointimal formation was evaluated. Genetic deletion of MFG-E8 in mice attenuated injury-induced neointimal hyperplasia. Cultured VSMCs deficient in MFG-E8 exhibited decreased cell migration. Immunofluorescence and immunoblotting revealed decreased Arp2 but not Arp3 expression in the common carotid arteries and VSMCs deficient in MFG-E8. Exogenous administration of recombinant MFG-E8 biphasically and dose-dependently regulated the cultured VSMCs. At a low concentration, MFG-E8 upregulated Arp2 expression. By contrast, MFG-E8 at a high concentration reduced the Arp2 level and significantly attenuated actin assembly. Arp2 upregulation mediated by low-dose MFG-E8 was abolished by treating cultured VSMCs with β1 integrin function-blocking antibody and Rac1 inhibitors. Moreover, treatment of the artery with a high dose of recombinant MFG-E8 diminished injury-induced neointimal hyperplasia and reduced VSMC migration. Conclusions MFG-E8 plays a critical role in VSMC migration through dose-dependent regulation of Arp2-mediated actin polymerization. These findings suggest that high doses of MFG-E8 may have therapeutic potential for treating vascular occlusive diseases.
Project description:OBJECTIVE:Bi-allelic function-inactivating ENPP1 mutations cause artery media calcification (AMC) with associated severe myointimal hyperplasia in generalized arterial calcification of infancy (GACI), whereas mono-allelic ENPP1 deficiency is phenotypically normal. Here, we tested if ENPP1 deficiency promotes abnormal vascular smooth muscle cell (VSMC)-driven responses to injury, with or without calcification. The ER stress mediator C/EBP homologous protein (CHOP) affects neointimal hyperplasia and atherosclerosis, and has paradoxical effects on bone formation. Hence, we assessed relationships between ENPP1 and CHOP in VSMCs. METHODS:We studied ENPP1-deficient mice and control littermates subjected to left carotid artery ligation, and isolated and studied VSMCs from these and Chop-/- mice, or with CHOP siRNA treatment. RESULTS:Normal Enpp1-/+ mice, in addition to Enpp1-/- mice prior to AMC development, had accelerated neointimal hyperplasia in response to carotid artery ligation at 7-8 weeks age. Neointimal hyperplasia was linked with robust artery media CHOP expression in situ, but with marked AMC only in injured Enpp1-/- arteries. Cultured, ENPP1-deficient and CHOP-deficient VSMCs had increased migration and proliferation to PDGF. Cultured Chop-/- VSMCs demonstrated increased Pi donor-induced calcification. CHOP was significantly increased in Pi donor treated Enpp1-/- and Enpp1-/+ cultured VSMCs. CHOP siRNA treatment of Enpp1-/- VSMCs increased calcification, associated with elevated expression of tissue nonspecific alkaline phosphatase and the master osteoblastic transcription factor RUNX2. CONCLUSIONS:Both mono-allelic and bi-allelic ENPP1 deficiency promote dysregulated VSMC function, with robust lesion CHOP expression and enhanced neointimal hyperplasia after injury in vivo, but marked post-injury calcification limited to Enpp1-/- mice. Intimal hyperplasia in GACI appears regulated by biologic effects of ENPP1 deficiency other than calcification, including ER stress. VSMC CHOP excess in ENPP1 deficiency may primarily function to limit VSMC calcification.