Mast cells (MCs) are progeny of multipotent hematopoietic stem cells that commit to the MC lineage already in the bone marrow. Although MCs are best known for their ability to release histamine and to induce IgE-mediated type I hypersensitivity reactions,1 they also initiate and regulate inflammatory responses, defend the host against bacterial and parasitic pathogens, regulate vascular functions, participate in wound healing and neovascularization, and recruit and activate other types of inflammatory cells and stromal cells as well.2,3 However, chronic local activation of MCs in diseases such as atherosclerosis,4 rheumatoid arthritis and congestive heart failure may result in a "foe" response that is unregulated, and if not properly terminated, may turn out to be harmful and even lethal to the host.
MCs contain a wide variety of preformed mediators that are secreted acutely upon MC activation with ensuing degranulation and that participate in the MC-mediated "friend or foe" responses.5 The preformed mediators can roughly be divided into five classes of effector molecules, notably histamine, proteoglycans, proteases, growth factors and cytokines, all of which may have an impact on the vulnerability of an atherosclerotic plaque.
Upon activation, MCs produce a plethora of different cytokines and chemokines that may actively participate in the pathogenesis of atherosclerotic plaques. These include at least the following: tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-β, interleukin (IL)-1α, IL-1β, IL-6, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF) and leucocyte inhibitory factor (LIF), all of which are involved in the induction of inflammation.6 Furthermore, the activated MCs also secrete IL-10 and transforming growth factor (TGF)-β, which are known to attenuate the inflammatory response in the atherosclerotic lesions.7
The activated MCs secrete proteases that either directly or indirectly can induce degradation of collagen, elastin and proteoglycans.8 Degradation of the extracellular and pericellular matrices induces apoptosis and weakens the fibrous cap making it more susceptible to rupture and precipitation of an acute coronary syndrome.
Compared with normal intima, the number of activated MCs in atherosclerotic plaques is especially high in the shoulder regions prone to plaque rupture.9 Interestingly, the level of MC degranulation was elevated in the intimal areas that contained an increased number of macrophages and T-lymphocytes, suggesting that factors responsible for MC degranulation in vivo may be derived from neighboring inflammatory cells.
MCs have also been shown to associate with neovessels formed within the atherosclerotic plaques.10 MCs, as a source of proangiogenic growth factors and proteases may also induce the formation of neovessels that traverse the medial layer into the intima and weaken the plaque, particularly when they rupture and cause intraplaque hemorrhage. Preventing the angiogenesis in plaque may play a very important role in stabilizing the vulnerable plaque.11
In addition, since the MCs present in the plaque are filled with the neutral proteases, tryptase, chymase and cathepsin G, which upon activation avidly secrete into the extracellular space, a therapeutic need to inhibit their activity in vivo may exist.
We have approached the mechanisms that MCs promote progression of atherosclerosis in cytology experiments, and have come to the following conclusions: (1) MCs improve oxidized low density lipoprotein induced smooth muscle cells (SMCs) foaming; and (2) MCs inhibit cholesterol efflux in THP-1 macrophage-derived foam cells.
To investigate the effects of MC degranulation on plaque and their possible mechanisms in animal experiments, we dealed with apolipoprotein E knockout (apoE-/-) mice which had been placed perivascular common carotid collar with MC degranulator compound 48–80. Compound 48–80 could stimulate MCs to degranulate and release active media such as histamine, tryptase, and chymase. The stain of toluidine blue and activity of tryptase were measured to demonstrate MC degranulation. In addition, we applied plaque area and the degree of lumen stenosis in the common carotid to evaluate effects of MC degranulation on plaque size. SMCs and macrophages were major cell components of plaque. Since it was debated whether MC degranulation promoted the proliferation of SMCs,12,13 we used immunohistochemistry to determine SMCs and macrophages in plaque. IL-1β is one of capital mediators of inflammation while inflammation is an important mechanism of progression of atherosclerosis. We adopted immunohistochemistry to determine expression of IL-1β in plaque to estimate effects of compound 48–80 on inflammatory response intensity. Expression of von Willebrand factor (vWF) and basic fibroblast growth factor (bFGF) in plaque were detected to further elucidate the underlying mechanisms of angiogenesis by MC degranulation in this model.
Male apoE-/- mice (Jackson Labs, USA) were ten-week-old at the time of entry into the study. The animals received a western-type diet (0.25 % cholesterol and 15 % lard) to the end of experiment.
Right common carotid collar placement
Collars were prepared from silastic tubing (Dow Corning, USA) and stored in 70% ethanol until use. Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg). The bilateral common carotids were dissected free from the surrounding connective tissue through a sagittal anterior neck incision. Silastic collar was placed around the right common carotid, and its axial edge was approximated by placement of 3 circumferential silk ties, which resulted in lumen stenosis. Subsequently, the entry wound was closed and the animal was returned to its cage for recovery from anesthesia.14
Four weeks after surgery, mice were divided into the following 2 groups (n=20) and treated for 7 days: experimental mice were intraperitoneally injected with compound 48–80 (Sigma, USA) 0.5 mg/kg, and control mice were intraperitoneally injected with an equal volume of D-Hanks every other day for 4 times.
Tissue harvesting and preparation
The mice were anesthetized and exsanguinated from the orbit after 30 minutes of the fourth injection with compound 48–80 or D-Hanks. Serum was collected for measurement of lipids and activity of tryptase. In situ perfusion fixation through the left cardiac ventricle was performed by PBS instillation for 15 minutes and followed by constant-pressure infusion (at 80 mmHg) of 10% neutral buffered formalin for 30 minutes. Subsequently, both common carotid arteries were removed, fixed in neutral buffered formalin, and embedded in paraffin.
Cholesterol and activity of tryptase assay
Blood samples were collected from the orbit. Total serum cholesterol was quantified by enzymatic procedures (Boehringer Mannheim, Germany) and the activity of tryptase was quantified by colorimetric assay (Sigma).
Histology and immunohistochemistry
Transverse 5 μm tissue sections were routinely stained with hematoxylin and eosin. Corresponding sections on separate slides were stained with toluidine blue (Sigma) and immunohistochemically with antibodies against macrophage-specific antigen (Mac3, polyclonal rat IgG, diluted 1:100; Santa Cruz, USA), α-smooth muscle actin (monoclonal mouse IgG, dilution 1:50, Wuhan Boshide, China), IL-1β (polyclonal rabbit IgG, diluted 1:50, Wuhan Boshide), and vWF (monoclonal rabbit IgG, diluted 1:100; Santa Cruz). The slides were incubated with primary antibody for 1 hour at room temperature or at 4°C overnight. Goat anti-rat IgG, goat anti-mouse IgG and goat anti-rabbit IgG peroxidase conjugate (dilution 1:100, BD, USA) were used as secondary antibodies (1 hour incubation at room temperature), with 3,3-diaminobenzidine as enzyme substrates.
Tissue sections on separate slides were stained with toluidine blue to quantify the percentage of degranulated MCs in the ectoblast of common carotids.
In situhybridization and immunofluorescence
Corresponding sections were stained with immunofluorescence (Beijing Zhongshanjinqiao, China) or in situ hybridization (Beijing Bolai, China) to detect the expression of bFGF in plaque.
Hematoxylin and eosin stained sections were used for morphometric analysis. The images were analyzed with an Olympus BX51 microscope and HMIAS2000 software. The plaque area was calculated by subtracting the patent lumen area from the area circumscribed by the internal elastic lamina. The degree of lumen stenosis was determined by plaque area/total lumen area ratio.
Results were expressed as mean ± standard deviation (SD) of the individual specimen measurements. Values of P <0.05 were considered statistically significant. Student's t test was used for comparison between the groups. The statistical analysis was done using SPSS version 12.0.
There were no significant difference in lipid levels between collar placement mice and sham-operated (non-collar placement) mice in the compound 48–80 group or the control group. But the activity of tryptase was increased in the compound 48–80 group (compound 48–80 group (0.57±0.13) U/L, control group (0.36±0.10) U/L, P <0.05).
Four weeks after surgery, no plaques were found in the corresponding sites of the common carotid arteries of the sham-operated mice of either group (Figure 1A). There were a significant increase in plaque area ((5.85±0.75) ×104μm2 vs (0.86±0.28) ×104 μm2, P <0.05) and the degree of lumen stenosis ((81±15)% vs (41±12)%, P <0.05) compared with the sham-operated in both groups (Figure 1B).
Percentage of degranulated MCs in the ectoblast of common carotids
The percentage of degranulated MCs in the ectoblast of common carotids was (80.6±17.8)% in the compound 48–80 group, and (13.5±4.1)% in the control group (P <0.05) (Figure 2).
The composition of the observed plaques was determined by a variety of immunohistochemical staining techniques. There were no significant differences in the composition of the observed plaques between the two groups. But the number of SMCs and macrophages of the observed plaques in the compound 48–80 group was more than that in the control group. The integral optical density of α-smooth muscle actin was (1219±364) vs (522±137) U (P <0.05), and the integral optical density of Mac3 was (426±133) vs (169±38) U (P <0.05) (Figure 3).
Angiogenesis in plaque
Sections on separate slides were stained immunohistochemically with antibody against vWF to count the density of neovessel in plaque and stained with immunofluorescence or in situ hybridization to detect the expression of bFGF in plaque in both groups. The density of neovessel in plaque was 859±278/mm2 in the compound 48–80 group, 366±97/mm2 in the control group, respectively (P <0.05). The integral optical density of bFGF mRNA was (926±291) U vs (213±63) U in the compound 48–80 group or in the control group (P <0.05), and the number of cells containing bFGF was 135±41 in the compound 48–80 group, and 47±18 in the control group, respectively (P <0.05) (Figure 4).
Expression of IL-1β in plaque
Sections were stained immunohistochemically with antibody against IL-1β to detect the expression of IL-1β in plaque in both groups. The integral optical density of IL-1β was (834±213) IU in the compound 48–80 group, and (189±57) IU in the control group (P <0.05) (Figure 5).
MCs are one of the inflammatory cells which participate in inflammation immune response. By secretion of pro-inflammatory cytokines, MCs can assist in the recruitment of monocytes and lymphocytes into vascular tissue, thereby propagating the inflammatory response. MC enzymes might activate pro-metalloproteinases, thereby destabilizing atheromatous plaques.15 Recent data from atherosclerotic patients and autopsied subjects have suggested that MCs might be important in the pathogenesis of atherosclerotic disease. Seventy-eight carotid samples from 75 patients (16 plaques from asymptomatic patients and 62 plaques from patients with recent ischemic symptoms) undergoing carotid endarterectomy with an internal carotid stenosis >70%. The samples were immunostained and quantified for MCs, macrophages and T cells. The average MC density other than the macrophage or T cell density was higher in the symptomatic than in the asymptomatic patients. Increased MC distribution density was found to be associated with high-grade carotid artery stenosis and symptomatic carotid artery disease.16 Atkinson et al17 studied the aortas and coronary arteries of 115 young subjects aged 15 to 34 years who died from trauma. Lesions were classified as normal intima, fatty streaks, fibro-fatty plaques, and fibrous plaques. Aortic and coronary artery segments with lesions had significantly a greater number of MCs in the adventitia (and occasionally intima and outer media) compared with those with a normal intima. In the aortic segments, a greater number of MCs were located in the dorsal portion (lesion "prone") compared with the ventral half (lesion "resistant") (P <0.05). These findings support the concept that an increased number of MCs are associated with atherosclerosis and a role for MC products in the evolution of the atherosclerotic plaque. Lappalainen's work revealed an increased number of bFGF-positive MCs in the neovascularized areas of human coronary plaques. The association of bFGF-positive MCs with microvessels and with the severity of atherosclerosis suggested that coronary MCs, by releasing bFGF, may play a role in angiogenesis and progression of coronary plaques.
In the present study we used apolipoprotein E knockout mice with perivascular common carotid collar placement as an animal model. The apolipoprotein E knockout mice known to spontaneously develop extensive and complex atherosclerotic lesions had hyperlipoidemia after feeding with a western-type diet. Placing a silastic collar (inside diameter of 0.3 mm ) on the mice resulted in a stenosis of 30% because the average outside diameter of the common carotid at 80 mmHg perfusion pressure was 0.36 mm, whereas the inside diameter of 0.5 mm was nonconstrictive (another experimental result of our study). Hence we suppose lumen stenosis caused by collar placement be important in formation of plaques.
We used compound 48–80 in the above-mentioned animal model to investigate the effects of MC degranulation on plaque and the possible mechanisms. The results demonstrated that compound 48–80 could increase the plaque area of maximum cross section, the degree of lumen stenosis, the number of SMCs and macrophages, the expression of IL-1β, angiogenesis in plaque.
Compound 48–80 increased the plaque area of maximum cross section and the degree of lumen stenosis probably by the mechanism that compound 48–80 promotes proliferation of SMCs and aggregation of macrophages. Some data indicated that chymase released by MC degranulation could transform angiotensin I into angiotensin II that promotes vascular smooth muscle cellular hypertrophy and/or hyperplasia depending i n part on the patterns of induction of secondary factors known to stimulate (plate-derived growth factor (PDGF), insulin-like growth factor (IGF)-1, bFGF) or inhibit TGF-β mitosis.18 The highest total angiotensin II-forming activity was observed in the heart and aorta of humans, and a chymaselike enzyme was dominant in all of the species except rabbit and pig, in which angiotensin converting enzyme (ACE) was dominant.19 Therefore, chymase inhibitor and angiotensin II receptor antagonist but angiotensin converting enzyme inhibitor (ACEI), ought to be employed to diminish the plaque area by refraining the proliferation of SMCs. Upon activation, MCs also secrete different chemotactic factors, for instance, macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and macrophage inflammatory protein-1β (MIP-1β). MCP-1, MIP-1α and MIP-1β, which belong to CC pattern chemotactic factor, promote peripheral blood mononuclear cells to immigrate into the intima and transform into macrophages, showing a significant event in the initial stage of atherosclerosis.
Angiogenesis is another important factor for the proliferation of SMCs in plaque, which provides an internal environment for growth and proliferation of SMCs. But in hypoxia, especially in the proliferation of SMCs, the hypoxia-inducible transcription factor (HIF) pathway is associated with plaque progression and angiogenesis.20
The aggregation of macrophages in plaque not only increases the plaque area but also secretes many mediators of inflammation such as IL-1β, IL-18, TNF-α, which play an important role in development of atherosclerosis. IL-1β can activate mononuclear/macrophage and lymphocyte to produce various cytokines and growth factors such as TNF-α, PDGF, IFN. It also can enhance the expression of adhesion molecules and induce the secretion of matrix metalloproteinase. 21 Thus, it is evident that aggregation of macrophages in the plaque induced by MCs can advance the development of the plaque through cascade effect of inflammation.
Compound 48–80 increased the expression of IL-1β in plaque in this experiment. We analyzed the cell source of IL-1β according to documents and found that it mostly comes from macrophage, MC, lymphocyte, SMCs, and endothelial cell. IL-1β possesses significant multitude biological effects, for instance, transcription and expression of PDGF which promote the proliferation of vascular SMC (VSMC) and vascular endothelial cell (VEC). These findings explain that compound 48–80 can advance proliferation and angiogenesis of SMCs in plaque.
Increased numbers of bFGF-positive MCs were detected in human coronary plaques. MCs of the intima and adventitia were a new cell source of bFGF in plaque. As a 18 kD basic polypeptide, bFGF was found to have chemotaxis to endothelial cell and directly stimulates endothelial cell to release basement membrane catabolic enzymes, migrate and proliferate, thus promoting neovascularization. Infusing bFGF onto the adventitia of the rat carotid artery contributed to the growth and maintenance of the vasa vasorum. The results of bFGF in situ hybridization and immunofluorescence in this study indicated that compound 48–80 increased expression of bFGF. We presumed that the cell source of bFGF was versatile on the basis of its diffuse distribution in plaque, including endothelial cell, SMCs, macrophage, MC, and so on. MC-derived tryptase degradated connective tissue matrix through activating collagenase and precursor of matrix metalloproteinase, accordingly provided space for angiogenesis and induced the formation of neovessels. 22
Promoting aggregation and activation of inflammatory cells is one of the mechanisms that MCs advance angiogenesis in plaque. Angiogenesis in plaque provides a passage way of inflammatory cells ingressing into plaque, then inflammatory cells secrete many cytokines and growth factors to promote angiogenesis.23 They form a vicious cycle to aggravate progression of atherosclerosis.
In summary, degranulation of MCs by compound 48–80 causes a significant increase in proliferation of SMCs, macrophage aggregation, IL-1β expression and angiogenesis in plaque. The latter four facilitate each other and advance the development of atherosclerosis. Probably it is a new idea for prevention and cure of atherosclerosis by the membrane stabilizer of MC.
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