Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

REVERSAL OF HELIX ORIENTATION IN THE CYANOBACTERIUM ARTHROSPIRA1

A survey of the morphological characters of 36 clonal axenic strains of Arthrospira showed that 34 had helical and 2 had straight trichomes. Of those with helical trichomes, five were right‐handed and 29 left‐handed. After repeated subculture for 1 year, the orientation of one helical strain (D893) had changed from right‐ to left‐handed, suggesting a probable genetic shift. The influence of environmental factors on helix orientation was tested on a subset of 10 strains. A temperature upshift from 30 to 32–34° C for 7 days led to a change in orientation in three strains (D918/H, D923, D925). Incubation at 32° C (D918/H, D923) or 34° C (D925) for periods less than needed for the morphological change to show still permitted the change to take place subsequently, when the temperature was reduced to 30° C; however, further subculture at 30° C led to the orientation reverting to its original state. In strain D925, but not the other nine strains, continuous shaking at 30° C also led to a change in helix orientation. In this case, some trichomes showed both orientations in a single trichome, with a snag at the point of reversion. A repeat survey of the stock cultures of all 34 strains after 2 years showed that another strain (D918/H) had now changed orientation from right‐handed to left‐handed. These observations are compared with the behavior of other helical structures in the literature, including filamentous Bacillus subtilis mutants and helix reversal in tendrils of climbing plants.     

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells

In smooth muscle cells (SMCs)isolated from rabbit carotid, femoral, and saphenous arteries, relativemyosin isoform mRNA levels were measured in RT-PCR to test forcorrelations between myosin isoform expression and unloaded shorteningvelocity. Unloaded shortening velocity and percent smooth muscle myosinheavy chain 2 (SM2) and myosin light chain 17b(MLC_17b) mRNA levels were not significantly different insingle SMCs isolated from the luminal and adluminal regions of thecarotid media. Saphenous artery SMCs shortened significantly faster(P < 0.05) than femoral SMCs and had more SM2 mRNA(P < 0.05) than carotid SMCs and lessMLC_17b mRNA (P < 0.001) and higher tissuelevels of SMB mRNA (P < 0.05) than carotid and femoralSMCs. No correlations were found between percent SM2 and percentMLC_17b mRNA levels and unloaded shortening velocity in SMCsfrom these arteries. We have previously shown that myosin heavy chain(MHC) SM1/SM2 and SMA/SMB and MLC_17a/MLC_17b isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression andunloaded shortening velocity do not vary with distance from the lumenof the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/orMLC_17a/MLC_17b isoform expression does notcorrelate with unloaded shortening velocity, and 3)intracellular expression of the MHC SM1/SM2 and MLC_17a/MLC_17b isoforms is not coregulated.

     

A theoretical and non-destructive experimental approach for direct inclusion of measured collagen orientation and recruitment into mechanical models of the artery wall

Gradual collagen recruitment has been hypothesized as the underlying mechanism for the mechanical stiffening with increasing stress in arteries. In this work, we investigated this hypothesis in eight rabbit carotid arteries by directly measuring the distribution of collagen recruitment stretch under increasing circumferential loading using a custom uniaxial (UA) extension device combined with a multi-photon microscope (MPM). This approach allowed simultaneous mechanical testing and imaging of collagen fibers without traditional destructive fixation methods. Fiber recruitment was quantified from 3D rendered MPM images, and fiber orientation was measured in projected stacks of images. Collagen recruitment was observed to initiate at a finite strain, corresponding to a sharp increase in the measured mechanical stiffness, confirming the previous hypothesis and motivating the development of a new constitutive model to capture this response. Previous constitutive equations for the arterial wall have modeled the collagen contribution with either abrupt recruitment at zero strain, abrupt recruitment at finite strain or as gradual recruitment beginning at infinitesimal strain. Based on our experimental data, a new combined constitutive model was presented in which fiber recruitment begins at a finite strain with activation stretch represented by a probability distribution function. By directly including this recruitment data, the collagen contribution was modeled using a simple Neo-Hookean equation. As a result, only two phenomenological material constants were required from the fit to the stress stretch data. Three other models for the arterial wall were then compared with these results. The approach taken here was successful in combining stress-strain analysis with simultaneous microstructural imaging of collagen recruitment and orientation, providing a new approach by which underlying fiber architecture may be quantified and included in constitutive equations.     

Caveolin-1 deficiency stimulates neointima formation during vascular injury

Neointima formation is a process characterized by smooth muscle cell (SMC) proliferation and extracellular matrix deposition in the vascular intimal layer. Here, we critically evaluate the role of caveolin-1 (Cav-1) in the pathogenesis of neointima formation. Cav-1 and caveolae organelles are particularly abundant in SMCs, where they are thought to function in membrane trafficking and signal transduction events. To directly evaluate the role of Cav-1 in the pathogenesis of neointimal lesions, we used Cav-1-deficient (Cav-1 -/-) mice as a model system. The right common carotid artery of wild-type and Cav-1 -/- mice was ligated just proximal to its bifurcation. Specimens were then harvested 4-weeks postligation and processed for morphometric and immunohistochemical analyses. The carotids of Cav-1 -/- mice showed significantly more intimal hyperplasia with subtotal luminal obstruction, as compared to wild-type mice. These neointimal lesions consisted mainly of SMCs. Mechanistically, neointimal lesions derived from Cav-1 -/- mice exhibited higher levels of phospho-p42/44 MAP kinase and cyclin D1 immunostaining, consistent with the idea that Cav-1 functions as a negative regulator of signal transduction. A significant increase in phospho-Rb (Ser780) immunostaining was also observed, in line with the upregulation of cyclin D1. In conclusion, using a carotid artery blood-flow cessation model, we show that genetic ablation of Cav-1 in mice stimulates SMC proliferation (neointimal hyperplasia), with concomitant activation of the p42/44 MAP kinase cascade and upregulation of cyclin D1. Importantly, our current study is the first to investigate the role of Cav-1 in SMC proliferation in the vascular system using Cav-1 -/- mice.     

Differential mechanical response and microstructural organization between non-human primate femoral and carotid arteries

Unique anatomic locations and physiologic functions predispose different arteries to varying mechanical responses and pathologies. However, the underlying causes of these mechanical differences are not well understood. The objective of this study was to first identify structural differences in the arterial matrix that would account for the mechanical differences between healthy femoral and carotid arteries and second to utilize these structural observations to perform a microstructurally motivated constitutive analysis. Femoral and carotid arteries were subjected to cylindrical biaxial loading and their microstructure was quantified using two-photon microscopy. The femoral arteries were found to be less compliant than the carotid arteries at physiologic loads, consistent with previous studies, despite similar extracellular compositions of collagen and elastin ( \(P> 0.05\) ). The femoral arteries exhibited significantly less circumferential dispersion of collagen fibers ( \(P< 0.05\) ), despite a similar mean fiber alignment direction as the carotid arteries. Elastin transmural distribution, in vivo axial stretch, and opening angles were also found to be distinctly different between the arteries. Lastly, we modeled the arteries’ mechanical behaviors using a microstructural-based, distributed collagen fiber constitutive model. With this approach, the material parameters of the model were solved using the experimental microstructural observations. The findings of this study support an important role for microstructural organization in arterial stiffness.     

PDGF-induced proliferation in human arterial and venous smooth muscle cells: molecular basis for differential effects of PDGF isoforms

Platelet‐derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum‐starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF‐AA, venous SMCs showed no proliferative response to PDGF‐AA, but instead demonstrated a significantly greater proliferative response to PDGF‐BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5‐fold higher (P < 0.05) density of PDGF receptor‐α (PDGF‐Rα) and a 6.6‐fold lower (P < 0.05) density of PDGF‐Rβ expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF‐AA in arterial SMCs was a marked PDGF‐Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c‐Jun NH2‐terminal kinase (JNK), and a significant reduction in expression of the cell‐cycle inhibitor p27^kip1. This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF‐Rα was detected after venous SMC exposure to PDGF‐AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF‐BB stimulation of venous SMC resulted in PDGF‐Rβ activation as well as transactivation of epidermal growth factor receptor (EGF‐R); transactivation of EGF‐R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins. J. Cell. Biochem. 112: 289–298, 2011. © 2010 Wiley‐Liss, Inc.     

Comparison of mechanical behavior among the extrapulmonary arteries from rats

Results of comparative tests on pulmonary arteries from untreated Long-Evans rats are presented from three sections of the artery: the trunk, and the right and left main extrapulmonary arteries. Analyses were conducted looking for mechanical differences between the flow (longitudinal) and circumferential directions, between the right and left main arteries, and between each of the mains and the trunk. The mechanical properties of rat pulmonary arteries were obtained with a bubble inflation technique. A flat disk of rat pulmonary artery was constrained at the periphery and inflated, and the geometry of the resulting bubble of material recorded from six different angles. To analyze the data, the area under the stress-strain curve was calculated for each test and orientation. This area, related to the strain-energy density, was calculated at stress equal to 200kPa, for the purpose of statistical comparison. The mean values for the area show that the trunk is less compliant than the main arteries; this difference is supported by histological evidence. When comparing the circumferential and longitudinal properties of the arteries, differences are found for the trunk and left main arteries, but with opposite orientations being more compliant. The mean values for the two orientations for the right main artery are statistically identical. There was indication of significant difference in mechanical properties between the trunk and the main arteries. The left main artery in the circumferential orientation is highly compliant and appears to strongly influence the likelihood that significant differences will exist when included in a statistical population. These data show that each section of the extrapulmonary arterial system should not be expected to behave identically, and they provide the baseline mechanical behavior of the pulmonary artery from normotensive rats.     

Medial and adventitial macrophages are associated with expansive atherosclerotic remodeling in rabbit femoral artery

Expansive vascular remodeling is considered a feature of vulnerable plaques. Although inflammation is upregulated in the media and adventitia of atherosclerotic lesions, its contribution to expansive remodeling is unclear. We investigated this issue in injured femoral arteries of normo- and hyperlipidemic rabbits fed with a conventional (CD group; n=20) or a 0.5% cholesterol (ChD group; n=20) diet. Four weeks after balloon injury of the femoral arteries, we examined vascular wall alterations, localization of macrophages and matrix metalloproteases (MMP)-1, -2, -9, and extracellular matrix. Neointimal formation with luminal stenosis was evident in both groups, while expansive remodeling was observed only in the ChD group. Areas immunopositive for macrophages, MMP-1, -2 and -9 were larger not only in the neointima, but also in the media and/or adventitia in the injured arterial walls of the ChD, than in the CD group. Areas containing smooth muscle cells (SMCs), elastin and collagen were smaller in the injured arterial walls of the ChD group. MMP-1, -2 and -9 were mainly localized in infiltrating macrophages. MMP-2 was also found in SMCs and adventitial fibroblasts. Vasa vasorum density was significantly increased in injured arteries of ChD group than in those of CD group. These results suggest that macrophages in the media and adventitia play an important role in expansive atherosclerotic remodeling via extracellular matrix degradation and SMC reduction.     

Proliferation of neointimal smooth muscle cells after arterial injury. Dependence on interactions between fibroblast growth factor receptor-2 and fibroblast growth factor-9

The growth factor signaling mechanisms responsible for neointimal smooth muscle cell (SMC) proliferation and accumulation, a characteristic feature of many vascular pathologies that can lead to restenosis after angioplasty, remain to be identified. Here, we examined the contribution of fibroblast growth factor receptors (FGFRs) 2 and 3 as well as novel fibroblast growth factors (FGFs) to such proliferation. Balloon catheter injury to the rat carotid artery stimulated the expression of two distinctly spliced FGFR-2 isoforms, differing only by the presence or absence of the acidic box, and two distinctly spliced FGFR-3 isoforms containing the acidic box and differing only by the presence of either the IIIb or IIIc exon. Post-injury arterial administration of recombinant adenoviruses expressing dominant negative mutant forms of these FGFRs were used to assess the roles of the endogenous FGFR isoforms in neointimal SMC proliferation. Dominant negative FGFR-2 containing the acidic box inhibited such proliferation by 40%, whereas the dominant negative FGFR-3 forms had little effect. Expression of FGF-9, known to be capable of binding to all four neointimal FGFR-2/-3 isoforms, was abundant within the neointima. FGF-9 markedly stimulated both the proliferation of neointimal SMCs and the activation of extracellular signal-related kinases 1/2, effects which were abrogated by the administration of antisense FGF-9 oligonucleotides to injured arteries and the expression of the dominant negative FGFR-2 adenovirus in cultured neointimal SMCs. These studies demonstrate that, although multiple FGFRs are induced in neointimal SMCs following arterial injury, specific interactions between distinctly spliced FGFR-2 isoforms and FGF-9 contribute to the proliferation of these SMCs.     

Flow pattern analysis in a highly stenotic patient-specific carotid bifurcation model using a turbulence model

The aim of this study is to investigate the blood flow pattern in carotid bifurcation with a high degree of luminal stenosis, combining in vivo magnetic resonance imaging (MRI) and computational fluid dynamics (CFD). A newly developed two-equation transitional model was employed to evaluate wall shear stress (WSS) distribution and pressure drop across the stenosis, which are closely related to plaque vulnerability. A patient with an 80% left carotid stenosis was imaged using high resolution MRI, from which a patient-specific geometry was reconstructed and flow boundary conditions were acquired for CFD simulation. A transitional model was implemented to investigate the flow velocity and WSS distribution in the patient-specific model. The peak time-averaged WSS value of approximately 73 Pa was predicted by the transitional flow model, and the regions of high WSS occurred at the throat of the stenosis. High oscillatory shear index values up to 0.50 were present in a helical flow pattern from the outer wall of the internal carotid artery immediately after the throat. This study shows the potential suitability of a transitional turbulent flow model in capturing the flow phenomena in severely stenosed carotid arteries using patient-specific MRI data and provides the basis for further investigation of the links between haemodynamic variables and plaque vulnerability. It may be useful in the future for risk assessment of patients with carotid disease.     

Role of cyclic strain frequency in regulating the alignment of vascular smooth muscle cells in vitro

The arterial system is subjected to cyclic strain because of periodic alterations in blood pressure, but the effects of frequency of cyclic strain on arterial smooth muscle cells (SMCs) remain unclear. Here, we investigated the potential role of the cyclic strain frequency in regulating SMC alignment using an in vitro model. Aortic SMCs were subject to cyclic strain at one elongation but at various frequencies using a Flexercell Tension Plus system. It was found that the angle information entropy, the activation of integrin-β1, p38 MAPK, and F/G actin ratio of filaments were all changed in a frequency-dependent manner, which was consistent with SMC alignment under cyclic strain with various frequencies. A treatment with anti-integrin-β1 antibody, SB202190, or cytochalasin D inhibited the cyclic strain frequency-dependent SMC alignment. These observations suggested that the frequency of cyclic strain plays a role in regulating the alignment of vascular SMCs in an intact actin filament-dependent manner, and cyclic strain at 1.25 Hz was the most effective frequency influencing SMC alignment. Furthermore, integrin-β1 and p38 MAPK possibly mediated cyclic strain frequency-dependent SMC alignment.     

Immunolocalization of matrix metalloproteinases in rabbit carotid arteries after balloon denudation

 Extracellular matrix-degrading enzymes may play a key role in vascular remodeling after arterial wall injury. We investigated the immunolocalization of matrix metalloproteinases (MMPs) in rabbit carotid arteries after balloon denudation. Positive immunostaining for MMP-1, -2, -3, and -9 appeared through the neointima 1 week after balloon denudation. The localization of immunopositive smooth muscle cells (SMCs) for MMP-1, -3, and -9, particularly for MMP-9, was almost similar to that of replicative SMCs and became confined to the luminal surface layer of the neointima at later time periods. However, MMP-2-positive SMCs appeared also in the basal layer of the neointima at 2 weeks, increased at 4 weeks, and then totally occupied the neointima at 6 weeks. The MMP-2-positive SMCs in the basal layer of the neointima at 4 and 6 weeks were negative for proliferation-associated antigens and were surrounded by extracellular matrix proteins. Our results suggest that all MMPs act in coordination to promote replication and migration of SMCs in the earlier phases of neointimal formation and that MMP-2 independently contributes to the later stages by facilitating the migration but not replication of SMCs from the media to the intima. Accepted: 25 June 1997     

兔颈动、静脉移植桥血管狭窄模型的建立及其电镜观察

目的：建立兔颈动、静脉移植血管桥动物模型,观察移植桥血管内膜增生和狭窄的电镜下表现。方法：通过兔双侧颈动脉进行动脉桥和静脉桥的移植,形成双侧移植血管桥再狭窄动物模型。在第8周施行血管桥移植手术的同时留取右侧颈动静脉标本作为对照血管,再分别于第12周、16周和第20周分别处死模型兔,采集移植桥血管标本,在光镜下测量其内膜厚度、面积、狭窄度,并进行电镜观察。结果：颈动脉和颈静脉桥移植后,随着时间的延长,桥血管的出现平滑肌迁移,脂质沉积,内膜增生,血管狭窄等改变,且以静脉桥血管的病理改变更为明显。结论：在兔形成动脉粥样硬化病变基础上,进行双侧颈动脉血管桥的移植,建立兔双侧颈动脉移植血管桥再狭窄动物模型,有利于设立自身对照,研究术后动静脉桥再狭窄差异机制;建立动、静脉桥后,位于血管中膜的平滑肌细胞出现向血管内膜迁移现象,说明中膜平滑肌细胞迁移进入内膜导致新内膜形成是血管再狭窄的重要环节。     

TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries

We tested the hypothesis that TRPC3, a member of the canonical transient receptor potential (TRP) family of channels, mediates agonist-induced depolarization of arterial smooth muscle cells (SMCs). In support of this hypothesis, we observed that suppression of arterial SMC TRPC3 expression with antisense oligodeoxynucleotides significantly decreased the depolarization and constriction of intact cerebral arteries in response to UTP. In contrast, depolarization and contraction of SMCs induced by increased intravascular pressure, i.e., myogenic responses, were not altered by TRPC3 suppression. Interestingly, UTP-evoked responses were not affected by suppression of a related TRP channel, TRPC6, which was previously found to be involved in myogenic depolarization and vasoconstriction. In patch-clamp experiments, UTP activated a whole cell current that was greatly reduced or absent in TRPC3 antisense-treated SMCs. These results indicate that TRPC3 mediates UTP-induced depolarization of arterial SMCs and that TRPC3 and TRPC6 may be differentially regulated by receptor activation and mechanical stimulation, respectively.     

Cytomegalovirus aggravates intimal hyperplasia in rats by stimulating smooth muscle cell proliferation

Epidemiological and animal studies suggest a role for cytomegalovirus (CMV) in restenosis. Previously, we demonstrated that proliferating smooth muscle cells (SMCs) in the injured arterial wall are particularly susceptible to CMV-induced effects. Therefore, we hypothesised that, depending on the time point of infection after vascular injury, CMV infection may affect cell proliferation either in the media or in the neointima, thereby aggravating the process of restenosis. In the present study, we focused on the individual layers of the arterial wall by evaluating, besides the neointima-to-media ratio, the medial and neointimal area and cellularity in the rat femoral artery. Vascular injury was photochemically induced in rat femoral arteries. Immediately or 14 days thereafter, rats were infected with rat CMV (RCMV) or mock infected. The presence of RCMV in the vascular wall was determined at 3, 5, 14 and 35 days after infection by quantitative real-time PCR. When rats were infected immediately after injury, a significant increase was seen only in the medial but not in the neointimal cross-sectional area. On the other hand, when rats were infected 14 days after the initial injury, a significant increase was only seen in the neointimal area, thereby confirming our hypothesis that RCMV infection primary affects proliferating SMCs. As the mean number of SMCs per microm2 in both cell layers was unchanged despite an increase in cross-sectional area, this implies that RCMV stimulated SMC proliferation. Furthermore, these vascular effects were observed without the virus being abundantly present in the vascular wall, suggesting that inflammatory and immune-mediated responses to RCMV infection are more important in aggravating the response to vascular injury than the virus itself.     

On the subclavian steal syndrome in vitro studies.

An elastic model of the arterial system has been used in which a specially designed pumping unit simulated the heart action. Physiological pressures and normal geometry, size, and flow distribution together with the normal cardiac output and use of prosthetic heart valves are the features of the system. Atherosclerosis was simulated by introducing blockages of known cross-section at specific sites of predilection. It has been shown that, for some specific occlusion magnitude in the left or right subclavian, or in the brachycephalic arteries, the stagnant no blood flow condition will appear in the left vertebral, or the right vertebral, or right common carotid, or the right internal carotid arteries. For larger occlusions the blood flow in these arteries reverses its direction, i.e., the "steal syndrome" appears. It is shown that besides the known single steal syndrome there exists also a double steal syndrome, i.e., blood reverses its flow direction simultaneously in two arteries, both on the right side of the arterial system. This blood is taken from the circle of Willis, which at the same time is significantly supplemented by the increased blood flow through the other arteries leading into the circle of Willis.     

Mechanical identification of layer-specific properties of mouse carotid arteries using 3D-DIC and a hyperelastic anisotropic constitutive model

The role of mechanics is known to be of primary order in many arterial diseases; however, determining mechanical properties of arteries remains a challenge. This paper discusses the identifiability of the passive mechanical properties of a mouse carotid artery, taking into account the orientation of collagen fibres in the medial and adventitial layers. On the basis of 3D digital image correlation measurements of the surface strain during an inflation/extension test, an inverse identification method is set up. It involves a 3D finite element mechanical model of the mechanical test and an optimisation algorithm. A two-layer constitutive model derived from the Holzapfel model is used, with five and then seven parameters. The five-parameter model is successfully identified providing layer-specific fibre angles. The seven-parameter model is over parameterised, yet it is shown that additional data from a simple tension test make the identification of refined layer-specific data reliable.