Activation of Smad2/3 signaling by low fluid shear stress mediates artery inward remodeling.

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-ß family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling.

circDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

Intimal hyperplasia is a reaction to vascular injury, which is the primary reason for vascular restenosis caused by the diagnostic or therapeutic procedure for cardiovascular diseases. Circular RNAs (circRNAs) are known to be associated with several cardiovascular conditions, but the expression of circRNAs in the neointima has not been reported in detail. In this study, we established the balloon-injured rat carotid artery model and detected the expression of circRNAs in the carotid arteries with a microarray. We found that the circRNA expression profile of the healthy carotid arteries and the injured arteries were significantly different. We investigated the role of rno-circ_005717 ( circDiaph3) in the differentiation of rat vascular smooth muscle cells (VSMCs). We found that knockdown of circDiaph3 up-regulated the level of diaphanous-related formin-3 and promoted the differentiation of VSMCs to contractile type. In addition, circDiaph3 up-regulated the transcription of Igf1r and supported the proliferation and migration of VSMCs. circDiaph3 could be a molecular target to combat intimal hyperplasia.-Xu, J.-Y., Chang, N.-B., Rong, Z.-H., Li, T., Xiao, L., Yao, Q.-P., Jiang, R., Jiang, J. circDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

Long noncoding RNA SNHG12 promotes vascular smooth muscle cell proliferation and migration via regulating miR-199a-5p/HIF-1α.

The dysregulation of proliferation and migration of vascular smooth muscle cells (VSMCs) contributes to atherosclerosis (AS) and accumulating reports indicate the crucial role of long noncoding RNA in AS. However, the role of small nucleolar RNA host gene 12 (SNHG12) in regulating the phenotypes of VSMCs and AS remains largely unknown. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of SNHG12 and miR-199a-5p in an in vivo AS model and VSMCs treated by oxidized low-density lipoprotein (ox-LDL). The proliferation ability, migration ability, and apoptosis of VSMCs were tested by cell counting kit-8, Transwell assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively. StarBase database was used to predict the binding sites between miR-199a-5p and SNHG12. The interaction between miR-199a-5p and SNHG12 was validated by qRT-PCR, western blot, and luciferase reporter assay. Western blot was used to examine the effects of SNHG12 and miR-199a-5p on the expression of hypoxia-inducible factor 1α (HIF-1α). We found that the expression level of SNHG12 was significantly increased in the animal model and VSMCs treated by ox-LDL. Knockdown of SNHG12 suppressed the proliferation and migration abilities of VSMCs, while overexpression of SNHG12 had the opposite effects. Mechanically, we validated that miR-199a-5p was a target of SNHG12, and the target gene of miR-199a-5p, HIF-1α could be indirectly and positively regulated by SNHG12. In conclusion, SHNG12 targeting miR-199a-5p/HIF-1α contributed to the pathophysiological process of AS by regulating the phenotypes of VSMCs, and could be a potential therapy target for this disease.

Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells.

Growth differentiation factor (GDF)11 has been reported to reverse age-related cardiac hypertrophy in mice and cause youthful regeneration of cardiomyocytes. The present study attempted to test a hypothesis that GDF11 counteracts the pathologic dedifferentiation of mouse carotid arterial smooth muscle cells (CASMCs) due to deficient autophagy. By real-time RT-PCR and Western blot analysis, exogenously administrated GDF11 was found to promote CASMC differentiation with increased expression of various differentiation markers (α-smooth muscle actin, myogenin, myogenic differentiation, and myosin heavy chain) as well as decreased expression of dedifferentiation markers (vimentin and proliferating cell nuclear antigen). Upregulation of the GDF11 gene by trichostatin A (TSA) or CRISPR-cas9 activating plasmids also stimulated the differentiation of CASMCs. Either GDF11 or TSA treatment blocked 7-ketocholesterol-induced CASMC dedifferentiation and autophagosome accumulation as well as lysosome inhibitor bafilomycin-induced dedifferentiation and autophagosome accumulation. Moreover, in CASMCs from mice lacking the CD38 gene, an autophagy deficiency model in CASMCs, GDF11 also inhibited its phenotypic transition to dedifferentiation status. Correspondingly, TSA treatment was shown to decrease GDF11 expression and reverse CASMC dedifferentiation in the partial ligated carotid artery of mice. The inhibitory effects of TSA on dedifferentiation of CASMCs were accompanied by reduced autophagosome accumulation in the arterial wall, which was accompanied by attenuated neointima formation in partial ligated carotid arteries. We concluded that GDF11 promotes CASMC differentiation and prevents the phenotypic transition of these cells induced by autophagosome accumulation during different pathological stimulations, such as Western diet, lysosome function deficiency, and inflammation. NEW & NOTEWORTHY The present study demonstrates that growth differentiation factor (GDF)11 promotes autophagy and subsequent differentiation in carotid arterial smooth muscle cells. Upregulation of GDF11 counteracts dedifferentiation under different pathological conditions. These findings provide novel insights into the regulatory role of GDF11 in the counteracting of sclerotic arterial diseases and also suggest that activation or induction of GDF11 may be a new therapeutic strategy for the treatment or prevention of these diseases.

Improved Patency of ePTFE Grafts as a Hemodialysis Access Site by Seeding Autologous Endothelial Cells Expressing Fibulin-5 and VEGF.

Small caliber synthetic vascular grafts used for dialysis access sites have high failure rates due to neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. We tested the use of ePTFE grafts seeded with autologous ECs expressing fibulin-5 and vascular endothelial growth factor (VEGF), as a dialysis access site in a porcine model. We connected the carotid arteries and jugular veins of 12 miniature pigs using 7-mm ePTFE grafts; five grafts were seeded with autologous venous ECs modified to express fibulin-5 and VEGF, and seven unseeded grafts were implanted at the same location and served as controls. At 6 months, after completion of angiography, the carotid arteries and jugular veins with the connecting grafts were excised and fixed. Autologous EC isolation and transduction and graft seeding were successful in all animals. At 3 months, 4 of 5 seeded grafts and 3 of 7 control grafts were patent. At 6 months, 4 of 5 (80%) seeded grafts and only 2 of 7 (29%) control grafts were patent. Seeding ePTFE vascular grafts with genetically modified ECs improved long term small caliber graft patency. The biosynthetic grafts offer a novel therapeutic modality for vascular access in hemodialysis.

Decellularization, cross-linking and heparin immobilization of porcine carotid arteries for tissue engineering vascular grafts.

Tissue engineering vascular grafts (TEVGs) have the potential to replace small-diameter grafts in bypass surgery which is good news for patients with cardiovascular disease. Decellularized arteries can be ideal TEVGs because their natural three-dimensional structures support the migration of host cells and vascular remodeling. There are many methods for decellularization without a standard protocol. In this study, a combination of Triton X-100 and sodium dodecyl sulfate (SDS) were used to prepare decellularized arteries. However, decellularization may damage the biochemical and mechanical properties to some degree. We used the cross-linking agents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to improve mechanical properties and immobilize heparin to inhibit thrombogenesis. Histological analysis, scanning electron microscopy, biomechanical properties test, determination of immobilized heparin, active partial thrombin time assay, and subcutaneous embedding experiment were used to evaluate the efficiency of decellularization and the efficacy of heparinized cross-linked vascular scaffold. Results showed 1% Triton X-100 combined with 0.3% SDS can decellularize successfully. EDC and NHS cross-linking can improve the mechanical properties, reduce the inflammatory reaction and slow the degradation time. Heparin immobilized on the scaffolds can inhibit thrombogenesis effectively. This study indicated the heparinized cross-linked vascular scaffolds may be ideal scaffolds for TEVGs.

Exosomes of Endothelial Progenitor Cells Inhibit Neointima Formation After Carotid Artery Injury.

BACKGROUND: Exosomes released from endothelial progenitor cells (EPCs) play a protective role in various disease models. Both endothelial cell (EC) damage and smooth muscle cell (SMC) proliferation are involved in the pathological process of restenosis after angioplasty and stenting. Few studies have focused on the therapeutic role of exosomes in EC damage and SMC proliferation. In this study, we sought to investigate the effect of exosomes released by human fetal aorta-derived EPCs on the rat carotid artery balloon injury model in vivo. We also sought to determine the effect of exosomes on both ECs and SMCs in vitro. METHODS: Exosomes (Exo group) or saline (Con group) were injected in rat carotid balloon injury model animals. The rats were sacrificed after 2, 4, 14, and 28 d, and injured carotid specimens were collected for Evans blue staining, hematoxylin-eosin staining, and immunohistochemistry. RESULTS: When the Con group and the Exo group were compared, the reendothelialized areas were not significantly different after 2 or 4 d, as shown by Evans blue staining. The hematoxylin-eosin results showed that the intimal to medial area ratio was slightly but not significantly higher in the Exo group after 2 and 4 d. The immunohistochemistry results showed that the proliferation of SMCs was slightly higher in the Exo group after 2 and 4 d, but the difference was not significant. The reendothelialization area of the Con group was significantly smaller than that of the Exo group at day 14. Both the intimal to medial area ratio and SMC proliferation in the Exo group were significantly smaller than those of the Con group at 14 or 28 d. In the in vitro study, exosome treatment significantly enhanced the proliferation and migration of both ECs and SMCs. CONCLUSIONS: Exosomes derived from EPCs could inhibit neointimal hyperplasia after carotid artery injury in rats. The protective effect of exosomes may manifest through the promotion of EC repair rather than direct suppression of proliferation and migration of smooth muscles cells.

Preparation and evaluation of decellularized porcine carotid arteries cross-linked by genipin: the preliminary results.

Decellularized arteries have been considered as promising scaffolds for small-diameter vascular substitutes. However, weakened mechanical properties, immunological rejection and rapid degradation after transplantation still exist after decellularization. Previous studies indicated that genipin cross-linking can solve these problems. Therefore, genipin was selected as the cross-linking agent for the pre-treatment of decellularized arteries in our study. Histological analysis, scanning electron microscopy, mechanical properties analysis and subcutaneous embedding experiment were adopted to investigate the efficiency of decellularization and the effect of genipin cross-linking on improving mechanical, structural and biological properties of decellularized arteries. Decellularization protocols based on three trypsin concentrations were used to prepare decellularized arteries, after decellularization, arteries were cross-linked with genipin. Results showed that 0.5% trypsin was the most efficient concentration to remove cellular components and preserve ECM. However, mechanical properties of 0.5% trypsin decellularized arteries weakened significantly, while genipin cross-linking improved mechanical properties of decellularized arteries to the same level as fresh arteries. After 4 weeks subcutaneous embedding, cross-linked arteries caused the mildest inflammatory response. In conclusion, genipin could be employed as an ideal cross-linking agent to strengthen mechanical properties, enhance the resistance to degradation and reduce the antigenicity of decellularized arteries for small-diameter blood vessel tissue engineering applications.

Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats.

Structural adaptation of arteries to weightlessness might lower the working ability or even threaten the physical health of astronauts, but the underlying mechanism is unclear. Acid sphingomyelinase (ASM) catalyzes ceramide (Cer) generation controlling arterial remodeling through multiple signaling pathways. In the present study, we aimed to investigate the contribution of ASM/Cer to the changes of common carotid artery intima-media thickness (CIMT) induced by simulated weightlessness. Hindlimb-unloaded tail-suspended (HU) rats were used to simulate the effect of weightlessness. Morphology of the carotid artery (CA) was examined by hematoxylin-eosin staining. Protein content of ASM or proliferating cell nuclear antigen (PCNA) was detected by Western blot. Cer level was measured by immunohistochemistry analysis. Apoptosis events were observed by transferase-mediated dUTP nick end labeling (TUNEL) staining. During 4 weeks of tail suspension, CIMT was increased gradually in HU but not in their synchronous control rats (P < 0.05). Correspondingly, the CA of HU rats had a lower apoptosis and higher proliferation of vascular smooth muscle cells (VSMCs). As compared to the control, both ASM protein expression and Cer content were reduced significantly in CA of HU rats (P < 0.05), incubation of which with permeable Cer reversed the changes in apoptosis and proliferation substantially. Furthermore, when the ASM protein content as well as Cer level in CA of control rats was diminished by using an ASM inhibitor, an increase of CIMT along with reduced apoptosis and enhanced proliferation of VSMCs was found. Our results suggest that by controlling the balance between apoptosis and proliferation, ASM/Cer plays an important role in the regulation of CIMT during simulated weightlessness.

Trimethylamine-N-Oxide Instigates NLRP3 Inflammasome Activation and Endothelial Dysfunction.

BACKGROUND/AIM: Plasma trimethylamine-N-oxide (TMAO), a product of intestinal microbial metabolism of dietary phosphatidylcholine has been recently associated with atherosclerosis and increased risk of cardiovascular diseases (CVD) in rodents and humans. However, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. The present study tested whether TMAO induces NLRP3 inflammasome formation and activation and thereby contributes to endothelial injury initiating atherogenesis. METHODS: Inflammasome formation and activation was determined by confocal microscopy, caspase-1 activity was measured by colorimetric assay, IL-1ß production was measured using ELISA, cell permeability was determined by microplate reader and ZO-1 expression was determined by western blot analysis and confocal microscopy. In in vivo experiments, TMAO was infused by osmotic pump implantation. RESULTS: TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase-1, caspase-1 activity, IL-1ß production, cell permeability in carotid artery endothelial cells (CAECs) compared to control cells. Pretreatment with caspase-1 inhibitor, WEHD or Nlrp3 siRNA abolished the TMAO-induced inflammasome formation, activation and cell permeability in these cells. In addition, we explored the mechanisms by which TMAO activates NLRP3 inflammasomes. TMAO-induced the activation of NLRP3 inflammasomes was associated with both redox regulation and lysosomal dysfunction. In animal experiments, direct infusion of TMAO in mice with partially ligated carotid artery were found to have increased NLRP3 inflammasome formation and IL-1ß production in the intima of wild type mice. CONCLUSION: The formation and activation of NLRP3 inflammasomes by TMAO may be an important initiating mechanism to turn on the endothelial inflammatory response leading to endothelial dysfunction.

Effects of DNA damage in smooth muscle cells in atherosclerosis.

RATIONALE: DNA damage and the DNA damage response have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double-stranded breaks (DSBs) are hypothesized to promote plaque progression and instability, in part, by promoting cell senescence, apoptosis, and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. OBJECTIVE: To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. METHODS AND RESULTS: Human atherosclerotic plaque VSMCs showed increased expression of multiple DNA damage response proteins in vitro and in vivo, particularly the MRE11/RAD50/NBS1 complex that senses DSB repair. Oxidative stress-induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated 2 novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with apolipoprotein E(-/-) mice. SM22α-NBS1/apolipoprotein E(-/-) VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/apolipoprotein E(-/-) VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. CONCLUSIONS: Human atherosclerotic plaque VSMCs show increased DNA damage, including DSBs and DNA damage response activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability.

Modeling the Effect of Red Blood Cells Deformability on Blood Flow Conditions in Human Carotid Artery Bifurcation.

The purpose of this work is to predict the effect of impaired red blood cells (RBCs) deformability on blood flow conditions in human carotid artery bifurcation. First, a blood viscosity model is developed that predicts the steady-state blood viscosity as a function of shear rate, plasma viscosity, and mechanical (and geometrical) properties of RBC's. Viscosity model is developed by modifying the well-known Krieger and Dougherty equation for monodisperse suspensions by using the dimensional analysis approach. With the approach, we manage to account for the microscopic properties of RBC's, such as their deformability, in the macroscopic behavior of blood via blood viscosity. In the second part of the paper, the deduced viscosity model is used to numerically predict blood flow conditions in human carotid artery bifurcation. Simulations are performed for different values of RBC's deformability and analyzed by investigating parameters, such as the temporal mean wall shear stress (WSS), oscillatory shear index (OSI), and mean temporal gradient of WSS. The analyses show that the decrease of RBC's deformability decrease the regions of low WSS (i.e., sites known to be prevalent at atherosclerosis-prone regions); increase, in average, the value of WSS along the artery; and decrease the areas of high OSI. These observations provide an insight into the influence of blood's microscopic properties, such as the deformability of RBC's, on hemodynamics in larger arteries and their influence on parameters that are known to play a role in the initiation and progression of atherosclerosis.

Annexin A1 Is a Physiological Modulator of Neutrophil Maturation and Recirculation Acting on the CXCR4/CXCL12 Pathway.

Neutrophil production and traffic in the body compartments is finely controlled, and the strong evidences support the role of CXCL12/CXCR4 pathway on neutrophil trafficking to and from the bone marrow (BM). We recently showed that the glucocorticoid-regulated protein, Annexin A1 (AnxA1) modulates neutrophil homeostasis and here we address the effects of AnxA1 on steady-state neutrophil maturation and trafficking. For this purpose, AnxA1(-/-) and Balb/C wild-type mice (WT) were donors of BM granulocytes and mesenchymal stem cells and blood neutrophils. In vivo treatments with the pharmacological AnxA1 mimetic peptide (Ac2-26) or the formyl peptide receptor (FPR) antagonist (Boc-2) were used to elucidate the pathway of AnxA1 action, and with the cytosolic glucocorticoid antagonist receptor RU 38486. Accelerated maturation of BM granulocytes was detected in AnxA1(-/-) and Boc2-treated WT mice, and was reversed by treatment with Ac2-26 in AnxA1(-/-) mice. AnxA1 and FPR2 were constitutively expressed in bone marrow granulocytes, and their expressions were reduced by treatment with RU38486. Higher numbers of CXCR4(+) neutrophils were detected in the circulation of AnxA1(-/-) or Boc2-treated WT mice, and values were rescued in Ac2-26-treated AnxA1(-/-) mice. Although circulating neutrophils of AnxA1(-/-) animals were CXCR4(+) , they presented reduced CXCL12-induced chemotaxis. Moreover, levels of CXCL12 were reduced in the bone marrow perfusate and in the mesenchymal stem cell supernatant from AnxA1(-/-) mice, and in vivo and in vitro CXCL12 expression was re-established after Ac2-26 treatment. Collectively, these data highlight AnxA1 as a novel determinant of neutrophil maturation and the mechanisms behind blood neutrophil homing to BM via the CXCL12/CXCR4 pathway. J. Cell. Physiol. 231: 2418-2427, 2016. © 2016 Wiley Periodicals, Inc.

Macrophages activate iNOS signaling in adventitial fibroblasts and contribute to adventitia fibrosis.

A large amount of NO is generated through the inducible nitric oxide synthase (iNOS) pathway from the vascular adventitia in various vascular diseases. However, it is currently not fully understood how the iNOS signaling pathway is activated. In the present study, this question was addressed in the context of adventitial cellular interactions. A rat model of acute hypertension in the contralateral carotid arteries was established through transverse aortic constriction (TAC) surgery. In this model, activated macrophages were found surrounded by a large quantity of iNOS-expressing adventitial fibroblasts (AFs), suggesting a possible causal relationship between macrophages and iNOS activation of the neighboring AFs. In an in vitro model, a macrophage-like cell line RAW 264.7 was first activated by LPS treatment. The supernatant was then harvested and applied to treat primary rat AFs. iNOS in AFs was activated robustly by the supernatant treatment but not by LPS itself. Treating AFs with interleukin-1ß (IL-1ß) also activated iNOS signaling, suggesting that the IL-1ß pathway might be a possible mediator. As a consequence of the iNOS activation, total protein nitration and S-nitrosylation significantly increased in those AFs. Additionally, increased deposition of type I and type III collagens was observed in both in vitro and in vivo models. The collagen deposition was partially restored by an iNOS inhibitor, 1400 W. These findings highlight the importance of iNOS signaling during vascular inflammation, and advance our understanding of its activation through a cellular interaction perspective.

Inhibition of four-and-a-half LIM domain protein-2 increases survival, migratory capacity, and paracrine function of human early outgrowth cells through activation of the sphingosine kinase-1 pathway: implications for endothelial regeneration.

RATIONALE: Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair. OBJECTIVE: We investigated the role of FHL2 in the regulation of EOCs. METHODS AND RESULTS: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell-derived factor-1-induced EOC migration through upregulation of αv/ß3, αv/ß5, and ß2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2(-/-) mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2(-/-) versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2(-/-) versus WT EOCs. CONCLUSIONS: Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.

Involvement of interleukin-1 receptor-associated kinase-1 in vascular smooth muscle cell proliferation and neointimal formation after rat carotid injury.

OBJECTIVE: Reduced frequency of atherosclerotic plaques is observed in interleukin-1 receptor-associated kinase-1 (IRAK1)-deficient mice; however, the underlying mechanism is not clear. Therefore, this study investigate the role of IRAK1 in vascular smooth muscle cell proliferation and neointimal hyperplasia. APPROACH AND RESULTS: Stimulation of rat primary vascular smooth muscle cells with fetal bovine serum (10%) or platelet-derived growth factor-BB (20 ng/mL) for 15 minutes to 24 hours induced a time-dependent increase in IRAK1 and extracellular signal-regulated kinase (ERK) activation, proliferating cell nuclear antigen upregulation and p27Kip1 downregulation as assessed by Western blotting. Inhibitors of ERK pathway (U0126, 10 µmol/L), IRAK (IRAK1/4, 3 µmol/L), protein kinase C (PKC; Ro-31-8220, 1 µmol/L), siRNA of toll-like receptor-4 (200 nmol/L), and PKC-ε (200 nmol/L) significantly attenuated these changes. Platelet-derived growth factor induced endogenous IRAK-ERK-PKC-ε association in a toll-like receptor-4 and PKC-ε-dependent manner. A time-dependent increase in IRAK1 and ERK activation was observed after 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours of carotid balloon injury in rats. Balloon injury induced endogenous IRAK-ERK-PKC-ε interaction. Perivascular application of IRAK1/4 inhibitor (100 µmol/L), U0126 (100 µmol/L), and IRAK1 siRNA (220 and 360 nmol/L) in pluronic gel abrogated balloon injury-induced ERK phosphorylation, activation, and p27Kip1 downregulation. Hematoxylin and eosin staining and immunohistochemistry of proliferating cell nuclear antigen and smooth muscle actin demonstrated that balloon injury-induced intimal thickening and neointimal vascular smooth muscle cell proliferation were significantly abrogated in the presence of IRAK1/4 inhibitor, IRAK1 siRNA, and U0126. CONCLUSIONS: IRAK1 mediates vascular smooth muscle cell proliferation and neointimal hyperplasia by regulating PKC-ε-IRAK1-ERK axis.

Embolus extravasation is an alternative mechanism for cerebral microvascular recanalization.

Cerebral microvascular occlusion is a common phenomenon throughout life that might require greater recognition as a mechanism of brain pathology. Failure to recanalize microvessels promptly may lead to the disruption of brain circuits and significant functional deficits. Haemodynamic forces and the fibrinolytic system are considered to be the principal mechanisms responsible for recanalization of occluded cerebral capillaries and terminal arterioles. Here we identify a previously unrecognized cellular mechanism that may also be critical for this recanalization. By using high-resolution fixed-tissue microscopy and two-photon imaging in living mice we observed that a large fraction of microemboli infused through the internal carotid artery failed to be lysed or washed out within 48 h. Instead, emboli were found to translocate outside the vessel lumen within 2-7 days, leading to complete re-establishment of blood flow and sparing of the vessel. Recanalization occurred by a previously unknown mechanism of microvascular plasticity involving the rapid envelopment of emboli by endothelial membrane projections that subsequently form a new vessel wall. This was followed by the formation of an endothelial opening through which emboli translocated into the perivascular parenchyma. The rate of embolus extravasation was significantly decreased by pharmacological inhibition of matrix metalloproteinase 2/9 activity. In aged mice, extravasation was markedly delayed, resulting in persistent tissue hypoxia, synaptic damage and cell death. Alterations in the efficiency of the protective mechanism that we have identified may have important implications in microvascular pathology, stroke recovery and age-related cognitive decline.

Platelet factor 4 mediates vascular smooth muscle cell injury responses.

Activated platelets release many inflammatory molecules with important roles in accelerating vascular inflammation. Much is known about platelet and platelet-derived mediator interactions with endothelial cells and leukocytes, but few studies have examined the effects of platelets on components of the vascular wall. Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to injury including the production of inflammatory molecules, cell proliferation, cell migration, and a decline in the expression of differentiation markers. In this study, we demonstrate that the platelet-derived chemokine platelet factor 4 (PF4/CXCL4) stimulates VSMC injury responses both in vitro and in vivo in a mouse carotid ligation model. PF4 drives a VSMC inflammatory phenotype including a decline in differentiation markers, increased cytokine production, and cell proliferation. We also demonstrate that PF4 effects are mediated, in part, through increased expression of the transcription factor Krüppel-like factor 4. Our data indicate an important mechanistic role for platelets and PF4 in VSMC injury responses both in vitro and in vivo.

MicroRNA-663 regulates human vascular smooth muscle cell phenotypic switch and vascular neointimal formation.

RATIONALE: Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation. OBJECTIVE: To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation. METHODS AND RESULTS: By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22α, smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor-induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. CONCLUSIONS: These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

Characterization of resident B cells of vascular walls in human atherosclerotic patients.

Animal models of atherosclerosis suggest that B cells have contradictory protective or proatherogenic effects that are also subset and context dependent. To further understand the pathophysiology of human atheroma, we characterized local Ig production and functional properties of resident B cells in human arterial lesions. Ig repertoires were analyzed by RT-PCR in carotid endarterectomy samples. Cytokine, differentiation marker and transcription factor mRNA expression was studied on arterial wall lymphocytes isolated by laser capture microdissection. Ig sequence analysis revealed that individual samples each contained a limited number of B cell clones. Functional α and γ mRNAs made up the majority of H chain mRNAs in the adventitia. Clonal evolution of Ig V regions, expression of activation-induced cytidine deaminase, clonal H chain switch, and an inverted λ/κ ratio of Ig L chain usage indicated that a local differentiation process was taking place in arterial walls. Clonotypic markers revealed different plaque and adventitia Ig repertoires and a B cell recirculation between adventitia and draining lymph nodes. Microdissected mononuclear cells had an activated phenotype expressing IL-6, GM-CSF, and TNF-α, whereas IL-2, IL-4, IL-10, M-CSF, and IFN-γ were not detected. Adventitial oligoclonal resident B cells of atherosclerotic patients are mainly mature B2 (conventional) CD20⁻ plasmablasts lacking markers of terminal differentiation to plasma cell (CD138 and Blimp-1). They present hallmarks of Ag-driven maturation and could act on inflammation and disease progression directly or by promoting polarization of other immune cells.