Medial Smooth Muscle Cells Research Articles

Peptide Inhibitor of CXCL4–CCL5 Heterodimer Formation, MKEY, Inhibits Experimental Aortic Aneurysm Initiation and Progression

Macrophages are critical contributors to abdominal aortic aneurysm (AAA) disease. We examined the ability of MKEY, a peptide inhibitor of CXCL4-CCL5 interaction, to influence AAA progression in murine models. AAAs were created in 10-week-old male C57BL/6J mice by transient infrarenal aortic porcine pancreatic elastase infusion. Mice were treated with MKEY via intravenous injection either (1) before porcine pancreatic elastase infusion or (2) after aneurysm initiation. Immunostaining demonstrated CCL5 and CCR5 expression on aneurysmal aortae and mural monocytes/macrophages, respectively. MKEY treatment partially inhibited migration of adaptively transferred leukocytes into aneurysmal aortae in recipient mice. Although all vehicle-pretreated mice developed AAAs, aneurysms formed in only 60% (3/5) and 14% (1/7) of mice pretreated with MKEY at 10 and 20 mg/kg, respectively. MKEY pretreatment reduced aortic diameter enlargement, preserved medial elastin fibers and smooth muscle cells, and attenuated mural macrophage infiltration, angiogenesis, and aortic metalloproteinase 2 and 9 expression after porcine pancreatic elastase infusion. MKEY initiated after porcine pancreatic elastase infusion also stabilized or reduced enlargement of existing AAAs. Finally, MKEY treatment was effective in limiting AAA formation after angiotensin II infusion in apolipoprotein E-deficient mice. MKEY suppresses AAA formation and progression in 2 complementary experimental models. Peptide inhibition of CXCL4-CCL5 interactions may represent a viable translational strategy to limit progression of human AAA disease.

Age-Related Variations of Caveolins Expression in the Wall of Saphenous Vein Used for Coronary Artery Bypass Grafting. A Preliminary Report

Background : Smooth muscle cells (SMCs) in the tunica media of the saphenous vein (SV) used for aortocoronary grafts play a key role in intimal hyperplasia. This process, followed by the development of atherosclerosis, leads to occlusion of the SV grafts months and years after coronary artery bypass grafting (CABG). Caveolins have been suggested to control some of the SMCs responses. The aim of this study was to identify any variations in expression of caveolins (cav-1, cav-2 and cav-3) in the SMCs of the SV transplants in relation to the age of CABG who undergo CABG. Methods : This study involved 110 (83 males and 27 females) consecutive, isolated and non-emergent CABG patients. They were retrospectively divided into four age subgroups: (A; n=19) ≤50 year-old, (B; n=24) >50 and ≤60 year-old, (C; n=40) >60 and ≤70 year-old, (D; n=27) >70 year-old. Expression of cav-1, cav-2 and cav-3 in the SV wall was evaluated by means of immunohistochemistry. Results : Expression of cav-1 and cav-3 in the SV wall increased systematically with the age of CABG patients and was statistically significant between group A and D (p<0.05). A positive correlation between the age of patients and expression of cav-1 and cav-3 was found (r=0.450 and r=0.463, respectively). Cav-2 expression did not differ between the examined subgroups. Conclusion : This study revealed positive correlations between the expressions of cav-1 and cav-3 in the medial venous SMCs and the age of CABG patients. However, further investigations are necessary to show clinical significance of these age-related variations.

Cathepsins: a new culprit behind abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a fatal disease defined as an abdominal aortic diameter of 3.0 cm or more, where the abdominal aorta exceeds the normal diameter by more than 50%. Histopathological changes of AAA mainly include extracellular matrix (ECM) remodeling at the abdominal aorta wall, but there is lack of specific drugs to treat AAA. Recent studies have reported that lysosomal cathepsins could induce vascular remodeling and AAA formation by regulating vascular inflammation, medial smooth muscle cell apoptosis, neovascularization, and protease expression. Thus, cathepsins are expected to become a new therapeutic target for AAA treatment.

Preferential secretion of collagen type 3 versus type 1 from adventitial fibroblasts stimulated by TGF-β/Smad3-treated medial smooth muscle cells

Restenosis, or arterial lumen re-narrowing, occurs in 30–50% of the patients undergoing angioplasty. Adaptive remodeling is the compensatory enlargement of the vessel size, and has been reported to prevent the deleterious effects of restenosis. Our previous studies have shown that elevated transforming growth factor (TGF-β) and its signaling protein Smad3 in the media layer induce adaptive remodeling of angioplastied rat carotid artery accompanying an increase of total collagen in the adventitia. In order to gain insights into a possible role of collagen in Smad3-induced adaptive remodeling, here we have investigated a mechanism of cell–cell communication between medial smooth muscle cells (SMCs) and adventitial fibroblasts in regulating the secretion of two major collagen subtypes. We have identified a preferential collagen-3 versus collagen-1 secretion by adventitial fibroblasts following stimulation by the conditioned medium from the TGF-β1-treated/Smad3-expressing medial smooth muscle cells (SMCs), which contained higher levels of CTGF and IGF2 as compared to control medium. Treating the TGF-β/Smad3-stimulated SMCs with an siRNA to either CTGF or IGF2 reversed the effect of conditioned media on preferential collagen-3 secretion from fibroblasts. Moreover, recombinant CTGF and IGF2 together stimulated adventitial fibroblasts to preferentially secrete collagen-3 versus collagen-1. This is the first study to identify a preferential secretion of collagen-3 versus collagen-1 from adventitial fibroblasts as a result of TGF-β/Smad3 stimulation of medial SMCs, and that CTGF and IGF2 function together to mediate this signaling communication between the two cell types.

Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection

BackgroundDescending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.MethodsWe studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.ResultsWestern blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.ConclusionsNotch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.

Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) pathway in experimental renal failure

Chronic renal failure (CRF) is associated with increased cardiovascular mortality, and medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification play a pivotal role in uremic vasculopathy. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear. This study aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression. In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed. Eight weeks after 5/6 nephrectomy, aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression. In vivo and in vitro experimental CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.

Age related structural changes in human basilar artery

The basilar artery is the most important artery in the posterior cerebral circulation. In the cases of stroke due to acute basilar artery occlusion the mortality rate is significantly higher if compared to all the stroke cases. Arterial wall stiffening is characteristic of ageing arteries and in many investigations arterial wall stiffening is related to the loss of the elastic component in the arterial wall during ageing. In arterial stiffening the changes in the collagen content and the number of smooth muscle cells (SMC) may also play a role. Basilar arteries were obtained within 24 hours postmortem from 89 human cadavers (44 male and 45 female). From the middle part of the basilar arteries histological slides were performed and area of collagen fibers, the number of smooth muscle cells and the thickness of the media were measured. The morphometric analysis revealed the increase of collagen network area with the age. Analysing age-related changes in the number of SMC in male and female basilar artery media, we determined that in both genders the number of SMC in the media decreased. Changes in SMC number in both genders had a strong negative correlation with the age (r=–0.93 in the male group and r=–0.95 in the female group, respectively). In the analysis of the media thickness in different gender and age groups, its thickening was determined. The correlation with the age was of medium strength both in the male (r=0.36) and female (r=0.4) group. In all the cases this correlation was statistically significant (p&lt;0.05). Our morphometric findings that revealed the increased collagen area together with the decrease of the SMC number might be responsible for the stiffening of the basilar artery in aging and contribute to the development of atherosclerosis and arterial hypertension.

Smooth muscle cells in human atherosclerosis: Proteomic profiling reveals differences in expression of Annexin A1 and mitochondrial proteins in carotid disease

Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The molecular mechanisms that mediate their properties are incompletely defined. We employed proteomics and in vitro functional assays to identify the unique characteristics of intimal SMC isolated from human carotid endarterectomy specimens and medial SMC from thoracic aortas and carotids. We verified our findings in the Tampere Vascular Study. Human atheroma-derived SMC exhibit decreased expression of mitochondrial proteins ATP Synthase subunit-beta and Aldehyde dehydrogenase 2, and decreased mitochondrial activity when compared to control SMC. Moreover, a comparison between plaque-derived SMC isolated from patients with or without recent acute cerebrovascular symptoms uncovered an increase in Annexin A1, an endogenous anti-inflammatory protein, in the asymptomatic group. The deletion of Annexin A1 or the blockade of its signaling in SMC resulted in increased cytokine production at baseline and after stimulation with the pro-inflammatory cytokine Tumor Necrosis Factor α. In summary, our proteomics and biochemical analysis revealed mitochondrial damage in human plaque-derived SMC as well as a role of Annexin A1 in reducing the production of pro-inflammatory mediators in SMC.

Coronary and Systemic Arterial Physiology and Immunohistochemical Markers Related to Early Coronary Arterial Lesions in Beagle Dogs Given the Potassium Channel Opener, ZD6169, or the Endothelin Receptor Antagonist, ZD1611

We evaluated immunohistochemistry (von Willebrand Factor [vWF] or fibrinogen) and systemic and coronary arterial physiological parameters in beagle dogs to investigate early arterial lesions induced by the potassium channel opener, ZD6169, or the endothelin receptor antagonist, ZD1611. Dogs given an oral dose of ZD6169 (experiment 1) were terminated 1 day later and showed arterial and myocardial lesions. Minimal arterial lesions exhibited few condensed medial smooth muscle cells only, with others showing segmental medial necrosis occasionally with medial/adventitial acute inflammation. Intercellular immunostaining was seen in ostensibly normal tissue, where no pathology was present in conventionally stained sections. vWF and fibrinogen are valuable tools for detecting disruption of arterial integrity. In experiment 2, 2 dogs were given a single high dose of ZD6169 or ZD1611 and BP/HR monitored by conventional measures or telemetry. Substantially reduced systolic/diastolic BP and increased HR occurred within 10 min of ZD6169 infusion: ZD1611 caused minor BP decrease and HR increase. In experiment 3, both drugs given to anaesthetized dogs induced markedly exaggerated systolic phasic forward and reverse flow in left descending and right coronary arteries. Diastolic coronary artery flows were unaffected with ZD1611 and increased slightly with ZD6169. In both coronary arteries, the ZD1611-induced increase in flows paralleled decreased resistance.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Association of smooth muscle cell phenotypes with extracellular matrix disorders in thoracic aortic dissection

Extracellular matrix dysregulation in the aortic media has been considered as the intrinsic factor for the formation of thoracic aortic dissection. However, the mechanisms of extracellular matrix disorders in the dissected aortic media remain unclear. This study was designed to investigate the relevance between smooth muscle cell phenotypes and extracellular matrix disorders in the dissected media. Their interaction may account for the pathogenesis of thoracic aortic dissection. Thoracic aortic samples were collected from 10 patients with thoracic aortic dissection and 10 controls. Primary cultures of aortic medial smooth muscle cells were obtained with optimized explant technique. In this study, α-smooth muscle actin, smooth muscle myosin heavy chain 2, and smoothelin were applied as the contractile phenotypic markers and osteopontin was applied as the synthetic marker. Compared with controls, immunostaining and immunoblotting demonstrated that in vivo expression of α-smooth muscle actin, smooth muscle myosin heavy chain 2, and smoothelin were significantly decreased in the dissected media, whereas that of osteopontin was elevated (P<.01 for all). In vitro expression of the phenotypic markers showed the similar patterns. Furthermore, smooth muscle cells derived from the dissected media exhibited enhanced proliferation (P<.01), increased collagens I and III synthesis (2.6- and 4.4-fold, respectively; P<.01 for both), and elevated matrix metalloproteinase-2 production (4.2-fold; P<.01). Consistently, the protein levels of type I and III collagens and matrix metalloproteinase-2 in the dissected media were raised by 4.6-, 4.0-, and 3.7-fold, respectively (P<.01 for all). Collagen deposition was correspondingly increased and elastic fibers were decreased and disrupted. Smooth muscle cells in the dissected media exhibit phenotypic switching from the contractile to the synthetic type. The synthetic smooth muscle cells increase collagen synthesis and matrix metalloproteinase-2 production, both of which can promote collagen deposition and elastin degradation in thoracic aortic dissection.

Low level laser arrests abdominal aortic aneurysm by collagen matrix reinforcement in apolipoprotein E‐deficient mice

Recent in vitro studies by our group indicated that low level laser irradiation (LLLI) modifies cellular processes essential to the progression of abdominal aortic aneurysm (AAA). Using high-frequency ultrasonography (HF-u/s) in the angiotensin-II (Ang-II)-infused, apolipoprotein-E-deficient (Apo-E(-/-) ) mouse model of AAA, we found that LLLI markedly inhibited aneurysm formation and preserved arterial wall elasticity. We now report, using quantitative histopathology, the likely mechanism underlying the preventative effect of LLLI on aneurysm formation in this model. This study was performed on 32 Apo-E(-/-) mice of which 10 were Ang-II-infused and LLL-irradiated (780 nm, 2 J/cm(2) , 9-minutes), 12 were Ang-II-infused but not irradiated, and 10 were saline infused. The aortas were excised at 28d, sectioned at 250 µm intervals, and stained with H + E, Movat-pentachrome and picrosirius-red for histomorphometry, and immunostained with Mac-2 and α-actin for detection of macrophages and SMCs, respectively. Transmural disruptions of the aorta occurred with distinct predilection for branch orifices. In the LLLI-treated animals, the frequency of these disruptions was lower (#branches with break points: 17 of 40 vs. 32 of 48, P = 0.023 by Chi-squared), their size smaller (length [mm]: 0.48 ± 0.26 vs. 0.98 ± 1.42, P = 0.044 by ANOVA with FPLSD), and the number of Mac-2-positive macrophages in the intramural areas of these disruptions lower than in the non-treated control (#Macrophages/0.01 mm(2) at break points: 11.6 ± 7.2 vs. 26.0 ± 15.7, P = 0.016 by Kruskal-Wallis). The average size of the medial SMCs was larger reflecting a heightened synthetic state (SMC size [µm(2) ]: 463.9 ± 61.4 vs. 354.9 ± 71.7, P = 0.001 by ANOVA with FPLSD). Furthermore, at sites of transmural disruption, the %area occupied by collagen of the overall area of attempted repair (%Col/WO) was significantly greater in the LLLI-treated animals versus control (%Col/WO: 41 ± 13 vs. 32 ± 16, P = 0.009 by ANOVA with FPLSD). Enhanced matrix reinforcement and modification of the inflammatory response at sites of transmural injury are prominent mechanisms by which LLLI reduces AAA progression in this model.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Chemokine (C-C motif) receptor 2 mediates mast cell migration to abdominal aortic aneurysm lesions in mice

Mast cells participate importantly in abdominal aortic aneurysms (AAAs) by releasing inflammatory cytokines to promote vascular cell protease expression and arterial wall remodelling. Mast cells accumulate in AAA lesions during disease progression, but the exact chemokines by which mast cells migrate to the site of vascular inflammation remain unknown. This study tested the hypothesis that mast cells use chemokine (C-C motif) receptor 2 (CCR2) for their accumulation in experimental mouse AAA lesions. We generated mast cell and apolipoprotein E double-deficient (Apoe(-/-)Kit(W-sh/W-sh)) mice and found that they were protected from angiotensin II (Ang II) chronic infusion-induced AAAs compared with Apoe(-/-) littermates. Using bone-marrow derived mast cells (BMMC) from Apoe(-/-) mice and CCR2 double-deficient (Apoe(-/-)Ccr2(-/-)) mice, we demonstrated that Apoe(-/-)Kit(W-sh/W-sh) mice receiving BMMC from Apoe(-/-)Ccr2(-/-) mice, but not those from Apoe(-/-) mice, remained protected from AAA formation. Adoptive transfer of BMMC from Apoe(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice also increased lesion content of macrophages, T cells, and MHC class II-positive cells; there was also increased apoptosis, angiogenesis, cell proliferation, elastin fragmentation, and medial smooth muscle cell loss. In contrast, adoptive transfer of BMMC from Apoe(-/-)Ccr2(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice did not affect these variables. The increased AAA formation and associated lesion characteristics in Apoe(-/-)Kit(W-sh/W-sh) mice after receiving BMMC from Apoe(-/-) mice, but not from Apoe(-/-)Ccr2(-/-) mice, suggests that mast cells use CCR2 as the chemokine receptor for their recruitment in Ang II-induced mouse AAA lesions.

The Cell Motility Modulator Slit2 Is a Potent Inhibitor of Platelet Function

Vascular injury and atherothrombosis involve vessel infiltration by inflammatory leukocytes, migration of medial vascular smooth muscle cells to the intimal layer, and ultimately acute thrombosis. A strategy to simultaneously target these pathological processes has yet to be identified. The secreted protein, Slit2, and its transmembrane receptor, Robo-1, repel neuronal migration in the developing central nervous system. More recently, it has been appreciated that Slit2 impairs chemotaxis of leukocytes and vascular smooth muscle cells toward diverse inflammatory attractants. The effects of Slit2 on platelet function and thrombus formation have never been explored. We detected Robo-1 expression in human and murine platelets and megakaryocytes and confirmed its presence via immunofluorescence microscopy and flow cytometry. In both static and shear microfluidic assays, Slit2 impaired platelet adhesion and spreading on diverse extracellular matrix substrates by suppressing activation of Akt. Slit2 also prevented platelet activation on exposure to ADP. In in vivo studies, Slit2 prolonged bleeding times in murine tail bleeding assays. Using intravital microscopy, we found that after mesenteric arteriolar and carotid artery injury, Slit2 delayed vessel occlusion time and prevented the stable formation of occlusive arteriolar thrombi. These data demonstrate that Slit2 is a powerful negative regulator of platelet function and thrombus formation. The ability to simultaneously block multiple events in vascular injury may allow Slit2 to effectively prevent and treat thrombotic disorders such as myocardial infarction and stroke.

Vascular Smooth Muscle Cell Apoptosis Promotes Transplant Arteriosclerosis Through Inducing the Production of SDF-1α

Transplant arteriosclerosis is a leading cause of late allograft loss. Medial smooth muscle cell (SMC) apoptosis is considered to be an important event in transplant arteriosclerosis. However, the precise contribution of medial SMC apoptosis to transplant arteriosclerosis and the underlying mechanisms remain unclear. We transferred wild-type p53 to induce apoptosis of cultured SMCs. We found that apoptosis induces the production of SDF-1α from apoptotic and neighboring viable cells, resulting in increased SDF-1α in the culture media. Conditioned media from Ltv-p53-transferred SMCs activated PI3K/Akt/mTOR and MAPK/Erk signaling in a SDF-1α-dependent manner and thereby promoted mesenchymal stem cell (MSC) migration and proliferation. In a rat aorta transplantation model, lentivirus-mediated BclxL transfer selectively inhibits medial SMC apoptosis in aortic allografts, resulting in a remarkable decrease of SDF-1α both in allograft media and in blood plasma, associated with diminished recruitment of CD90(+)CD105(+) double-positive cells and impaired neointimal formation. Systemic administration of rapamycin or PD98059 also attenuated MSC recruitment and neointimal formation in the aortic allografts. These results suggest that medial SMC apoptosis is critical for the development of transplant arteriosclerosis through inducing SDF-1α production and that MSC recruitment represents a major component of vascular remodeling, constituting a relevant target and mechanism for therapeutic interventions.

Osteoblast-like Differentiation of Cultured Human Coronary Artery Smooth Muscle Cells by Bone Morphogenetic Protein Endothelial Cell Precursor-derived Regulator (BMPER)

Differentiation of vascular smooth muscle cells (SMCs) into osteoblast-like cells is considered to be a mechanism of vascular calcification. However, regulators of osteoblast-like differentiation of vascular SMCs are not fully elucidated. Here, we investigated the expression of bone morphogenetic protein (BMP)-binding endothelial cell precursor-derived regulator (BMPER), a vertebrate homologue of Drosophila crossveinless-2, in vascular SMCs and the role and mode of action of BMPER in osteoblast-like differentiation of human coronary artery SMCs (HCASMCs). BMPER was expressed in cultured human vascular SMCs, including HCASMCs. Silencing of endogenous BMPER expression by an RNA interference technique inhibited osteoblast-like differentiation of HCASMCs, as evaluated by up-regulation of osteoblast markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), by down-regulation of a SMC marker α-smooth muscle actin (αSMA), and by mineralization. Treatment with recombinant BMPER enhanced, whereas BMP-2 reduced osteoblast-like differentiation. BMPER antagonized BMP-2-induced phosphorylation of Smad 1/5/8, suggesting that the effect of BMPER was mediated by antagonizing the action of BMP. BMPER increased IκBα phosphorylation and NF-κB activity and specific NF-κB decoy oligonucleotides deteriorated osteoblast-like differentiation of HCASMCs by BMPER. In human coronary artery with atherosclerotic plaque containing calcification, the BMPER-positive signals were observed in the neointimal and medial SMCs in the vicinity of the plaque. These findings indicate that BMPER is a novel regulator of the osteoblast-like differentiation of HCASMCs.

Ultrastructural Changes of Smooth Muscles in Varicocele Veins

Background: Varicocele is a dilatation of the pampiniform venous plexus and internal spermatic veins. It affects about 15–20% of male population and can cause infertility.Objective: To describe the most significant ultrastructural changes of the smooth muscle cells in grade 3 varicocele veins.Methods: The authors analyzed 2- to 3-cm tracts of pampiniform venous plexus from 20 patients who underwent varicocelectomy for left varicocele. Light microscopic examination was performed with Van Gieson’s stain. Ultrastructural examination was done using scanning and transmission electron microscopy.Results: Light microscopic examination revealed irregularity and separation of medial smooth muscle cells by abundant collagen fibers in varicocele veins. On scanning electron microscopy, the medial layer of varicocele veins showed hypertrophy, irregularity, and separation of the outer longitudinal smooth muscle cells and deposition of numerous fatty globules in between muscle fibers. Transmission electron microscopy showed marked indentation and chromatin condensation of the nucleus, presence of clear vacuoles and myelin figures in the cytoplasm and plasmalemmal projections and formation of ghost bodies. Furthermore, smooth muscle cells were found to have pseudopodia-like projections around adjacent elastic and collagen fibers.Conclusions: The degenerative changes observed in smooth muscle cells and presence of abundant collagen fibers in the medial layer may contribute to the development of the varicocele of pampiniform venous plexus. Further molecular studies are required to shed more light for the underlying mechanism.

Endothelial Lipase Is Localized to Follicular Epithelial Cells in the Thyroid Gland and Is Moderately Expressed in Adipocytes

Endothelial lipase (EL), a member of the triglyceride lipase gene family, has been shown to be a key player in HDL metabolism. Northern blots revealed that EL was highly expressed in endothelium, thyroid, lung, placenta, liver, and testis. In liver and adrenal gland, EL protein was localized with vascular endothelial cells but not parenchymal cells. EL was shown to be upregulated in tissues such as atherosclerotic plaque where it was located in macrophages, endothelial cells, and medial smooth muscle cells. The purpose of this study was to investigate the cellular localization of EL in thyroid and other tissues where EL is known to be expressed. Besides its presence in vascular endothelial and smooth muscle cells, EL protein was detected in the epithelial cells that line the follicles within the thyroid gland. EL-specific immunostaining was also found near the cell surface as well as in the cytoplasm of adipocytes. Using immunoblots, EL expression was confirmed in cultured human omental and subcutaneous adipocytes. EL expression, however, was not found in preadipocytes. These findings suggest that EL plays a role in thyroid and adipocyte biology in addition to its well-known role in endothelial function and HDL metabolism.

Three Linked Vasculopathic Processes Characterize Kawasaki Disease: A Light and Transmission Electron Microscopic Study

BackgroundKawasaki disease is recognized as the most common cause of acquired heart disease in children in the developed world. Clinical, epidemiologic, and pathologic evidence supports an infectious agent, likely entering through the lung. Pathologic studies proposing an acute coronary arteritis followed by healing fail to account for the complex vasculopathy and clinical course.Methodology/Principal FindingsSpecimens from 32 autopsies, 8 cardiac transplants, and an excised coronary aneurysm were studied by light (n=41) and transmission electron microscopy (n=7). Three characteristic vasculopathic processes were identified in coronary (CA) and non-coronary arteries: acute self-limited necrotizing arteritis (NA), subacute/chronic (SA/C) vasculitis, and luminal myofibroblastic proliferation (LMP). NA is a synchronous neutrophilic process of the endothelium, beginning and ending within the first two weeks of fever onset, and progressively destroying the wall into the adventitia causing saccular aneurysms, which can thrombose or rupture. SA/C vasculitis is an asynchronous process that can commence within the first two weeks onward, starting in the adventitia/perivascular tissue and variably inflaming/damaging the wall during progression to the lumen. Besides fusiform and saccular aneurysms that can thrombose, SA/C vasculitis likely causes the transition of medial and adventitial smooth muscle cells (SMC) into classic myofibroblasts, which combined with their matrix products and inflammation create progressive stenosing luminal lesions (SA/C-LMP). Remote LMP apparently results from circulating factors. Veins, pulmonary arteries, and aorta can develop subclinical SA/C vasculitis and SA/C-LMP, but not NA. The earliest death (day 10) had both CA SA/C vasculitis and SA/C-LMP, and an “eosinophilic-type” myocarditis.Conclusions/SignificanceNA is the only self-limiting process of the three, is responsible for the earliest morbidity/mortality, and is consistent with acute viral infection. SA/C vasculitis can begin as early as NA, but can occur/persist for months to years; LMP causes progressive arterial stenosis and thrombosis and is composed of unique SMC-derived pathologic myofibroblasts.