Contribution Of Smooth Muscle Cells Research Articles

Smooth muscle Cxcl12 activation is associated with vascular remodeling in flow-induced pulmonary hypertension.

Patients with congenital heart disease (CHD) resulting in significant left-to-right shunting of blood are at risk for the development of pulmonary arterial hypertension (PAH). The underlying mechanism by which pulmonary overcirculation and shear stress lead to vascular remodeling remains unclear. Our study established a new "two-hit" murine model of severe pulmonary hypertension (PH) by combining left pneumonectomy and exposure to hypoxia (LP/Hx). Utilizing transgenic reporter lines, immunofluorescence staining, and advanced microscopy, we conducted cell-lineage tracing experiments for endothelial cells (ECs), smooth muscle cells (SMCs), and pericytes. We identified that SMCs is a primary contributor to distal arteriolar remodeling after LP/Hx. Subsequent qPCR analysis on isolated cells demonstrated that Cxcl12 was upregulated in both ECs and SMCs from LP/Hx animals. Likewise, CXCL12 was overexpressed in the SMC layer of arterioles in patients with acyanotic PAH-CHD. These findings provide novel insights into the contribution of SMCs and Cxcl12 to pulmonary flow-induced vascular remodeling. This newly established murine model of PH will serve as a new tool for research and targeted therapeutics for patients with PAH.

Remodeling of murine vaginal smooth muscle function with reproductive age and elastic fiber disruption

Advanced maternal age during pregnancy is associated with increased risk of vaginal tearing during delivery and maladaptive postpartum healing. Although the underlying mechanisms of age-related vaginal injuries are not fully elucidated, changes in vaginal microstructure may contribute. Smooth muscle cells promote the contractile nature of the vagina and contribute to pelvic floor stability. While menopause is associated with decreased vaginal smooth muscle content, whether contractile changes occur before the onset of menopause remains unknown. Therefore, the first objective of this study was to quantify the active mechanical behavior of the murine vagina with age. Further, aging is associated with decreased vaginal elastin content. As such, the second objective was to determine if elastic fiber disruption alters vaginal contractility. Vaginal samples from mice aged 2–14 months were used in maximum contractility experiments and biaxial extension-inflation protocols. To evaluate the role of elastic fibers with age, half of the vaginal samples were randomly allocated to enzymatic elastic fiber disruption. Contractile potential decreased and vaginal material stiffness increased with age. These age-related changes in smooth muscle function may be due, in part, to changes in microstructural composition or contractile gene expression. Furthermore, elastic fiber disruption had a diminished effect on smooth muscle contractility in older mice. This suggests a decreased functional role of elastic fibers with age. Quantifying the age-dependent mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties provides a first step towards better understanding how age-related changes in vaginal structure may contribute to tissue integrity and healing. Statement of significanceAdvanced maternal age at the time of pregnancy is linked to increased risks of vaginal tearing during delivery, postpartum hemorrhaging, and the development of pelvic floor disorders. While the underlying causes of increased vaginal injuries with age and associated pathologies remain unclear, changes in vaginal microstructure, such as elastic fibers and smooth muscle cells, may contribute. Menopause is associated with fragmented elastic fibers and decreased smooth muscle content; however, how reproductive aging affects changes in the vaginal composition and the mechanical properties remains unknown. Quantifying the mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties with age will advance understanding of the potential structural causes of age-related changes to tissue integrity and healing.

A single-cell atlas of the myometrium in human parturition.

Parturition is a well-orchestrated process characterized by increased uterine contractility, cervical ripening, and activation of the chorioamniotic membranes; yet, the transition from a quiescent to a contractile myometrium heralds the onset of labor. However, the cellular underpinnings of human parturition in the uterine tissues are still poorly understood. Herein, we performed a comprehensive study of the human myometrium during spontaneous term labor using single-cell RNA sequencing (scRNA-Seq). First, we established a single-cell atlas of the human myometrium and unraveled the cell type–specific transcriptomic activity modulated during labor. Major cell types included distinct subsets of smooth muscle cells, monocytes/macrophages, stromal cells, and endothelial cells, all of which communicated and participated in immune (e.g., inflammation) and nonimmune (e.g., contraction) processes associated with labor. Furthermore, integrating scRNA-Seq and microarray data with deconvolution of bulk gene expression highlighted the contribution of smooth muscle cells to labor-associated contractility and inflammatory processes. Last, myometrium-derived single-cell signatures can be quantified in the maternal whole-blood transcriptome throughout pregnancy and are enriched in women in labor, providing a potential means of noninvasively monitoring pregnancy and its complications. Together, our findings provide insights into the contributions of specific myometrial cell types to the biological processes that take place during term parturition.

Efficient computational modelling of smooth muscle orientation and function in the aorta

Understanding the mechanical effects of smooth muscle cell (SMC) contraction on the initiation and the propagation of cardiovascular diseases such as aortic dissection is critical. Framed by elastic lamellar sheets in the lamellar unit, there are SMCs in the media with a distinct radial tilt, which indicates their contribution to the radial strength. However, the mechanical effects of this type of anisotropy have not been fully discussed. Therefore, in this study, we propose a constitutive framework that models the passive and active mechanics of the aorta, taking into account the dispersed nature of the aortic constituents by applying the discrete fibre dispersion method. We suggest an isoparametric approach by evaluating various numerical integration methods and introducing a non-uniform discretization of the unit hemisphere to increase its computational efficiency. Finally, the constitutive parameters are fitted to layer-specific experimental data and initial computational results are briefly presented. The radial tilt of SMCs is also analysed, which has a noticeable influence on the mechanical behaviour of the aorta. In the absence of sufficient experimental data, the results indicate that the active contribution of SMCs has a remarkable impact on the mechanics of the healthy aorta.

Evaluation of the effect of smooth muscle cells on the quality of cultured meat in a model for cultured meat

While the research on improving the meat quality of cultured meat is in full swing, few studies have focused on the effect of smooth muscle cells (SMCs) on the meat quality of cultured meat. Therefore, this study aimed at building a cultured meat model containing smooth muscle cells, and further evaluating the effect of smooth muscle cells on the quality of cultured meat, so as to reveal the contribution of smooth muscle cells in the production of cultured meat. In this study, we isolated high purity of smooth muscle cells from vascular tissues. The addition of basic fibroblast growth factor (bFGF) to the medium significantly increased the growth rate of smooth muscle cells and the expression of extracellular matrix related genes, especially collagen and elastin. Smooth muscle cells were seeded in a collagen gel to construct a culture meat model. It was found that the pressure loss of the model meat significantly decreased from 98.5 % in control group to 54 % with the extension of culture time for 9 days, while the total collagen content of model meat increased significantly (P < 0.05). In addition, the hydrogel tissue with smooth muscle cells compacted more dramatically and were more tightly, accompanied by significantly increased hardness, springiness and chewiness compared to the control one (P < 0.05). These results indicate that smooth muscle cells can secrete extracellular matrix proteins such as collagen, which can significantly enhance the texture of cultured meat models prepared by hydrogel.

Tissue-engineered arterial intima model exposed to steady wall shear stresses

The arterial intima is continuously under pulsatile wall shear stresses (WSS) imposed by the circulating blood. The knowledge of the contribution of smooth muscle cells (SMC) to the response of endothelial cell (EC) to WSS is still incomplete. We developed a co-culture model of EC on top of SMC that mimics the inner in vivo structure of the arterial intima of large arteries. The co-cultured model, as well as a monolayer model of EC, were developed in custom-designed wells that allowed for mechanobiology experiments. Both the monolayer and co-culture models were exposed to steady flow induced WSS of up to 24 dyne/cm2 and for lengths of 60 min. Quantification of WSS induced alterations in the cytoskeletal actin filaments (F-actin) and vascular endothelial cadherin (VE-cadherin) junctions were utilized from confocal images and flow cytometry. High confluency of both models was observed even after exposure to the high WSS. The quantitive analysis revealed larger post WSS amounts of EC F-actin polymerization in the monolayer, which may be explained by the relative help of the SMC to resist the external load of WSS. The VE-cadherin demonstrated morphological alterations in the monolayer model, but without significant changes in their content. The SMC in the co-culture maintained their contractile phenotype post high WSS which is more physiological, but not post low WSS. Generally, the results of this work demonstrate the active role of SMC in the intima performance to resist flow induced WSS.

Endothelial Dysfunction and Passive Changes in the Aorta and Coronary Arteries of Diabetic db/db Mice.

Endothelial cell dysfunction and vessel stiffening are associated with a worsened prognosis in diabetic patients with cardiovascular diseases. The present study hypothesized that sex impacts endothelial dysfunction and structural changes in arteries from diabetic mice. In diabetic (db/db) and normoglycaemic (db/db+) mice, the mechanical properties were investigated in pressurized isolated left anterior descending coronary arteries and aorta segments that were subjected to tensile testing. Functional studies were performed on wire-mounted vascular segments. The male and female db/db mice were hyperglycaemic and had markedly increased body weight. In isolated aorta segments without the contribution of smooth muscle cells, load to rupture, viscoelasticity, and collagen content were decreased suggesting larger distensibility of the arterial wall in both male and female db/db mice. In male db/db aorta segments with smooth muscle cell contribution, lumen diameter was smaller and the passive stretch-tension curve was leftward-shifted, while they were unaltered in female db/db aorta segments versus control db/db+ mice. In contrast to female db/db mice, coronary arteries from male db/db mice had altered stress-strain relationships and increased distensibility. Transthoracic echocardiography revealed a dilated left ventricle with unaltered cardiac output, while aortic flow velocity was decreased in male db/db mice. Impairment of acetylcholine relaxation was aggravated in aorta from female db/db compared to control and male db/db mice, while impairment of sodium nitroprusside relaxations was only observed in aorta from male db/db mice. The remodeling in the coronary arteries and aorta suggests an adaptation of the arterial wall to the reduced flow velocity with sex-specific differences in the passive properties of aorta and coronary arteries. The findings of less distensible arteries and more pronounced endothelial dysfunction in female compared to male diabetic mice may have implications for the observed higher incidence of macrovascular complications in diabetic women.

Non-axisymmetric dilatation of a thick-walled aortic aneurysmal tissue

The objective of this paper is to use the constrained mixture theory of growth and remodeling to simulate the non-axisymmetric dilatation of a thick-walled aortic aneurysmal tissue. The primary load carrying constituents of the vascular tissue are elastin and collagen and the contribution of smooth muscle cells is secondary and therefore not included. In the homeostatic state a blood vessel is in a mechanobiologically stable regime. Hence, a loss of wall material is compensated by production of new material without a significant dilatation of the artery. Using the theory we find that a local degradation of the matrix material produces a mechanobiologically unstable regime that causes aneurysm formation. It induces an increase of mass locally achieved via production of new material that exceeds the removal of old material. The combined effects of loss of elastin, degradation of existing and deposition of new collagen as well as remodeling results in a continuous enlargement of the aneurysm bulge. Numerical results are included to verify and validate the theory.

Abstract 640: Genetic Inactivation of Col15a1 in Smooth Muscle Cells Decreases Plaque Size and Decreases Indices of Plaque Stability

In the development of atherosclerosis, it is widely believed that contractile smooth muscle cells (SMC) increase their migratory and proliferative capacity and invade the neointima where they secrete extracellular matrix (ECM) to form a plaque stabilizing fibrous cap. We previously identified that collagen, type XV, alpha 1 (COL15A1) expression is increased in human atherosclerotic plaques and associated with lesion burden. COL15A1 has been shown to link large fibrillar collagens to confer ECM stability. Of interest, siRNA knockdown of Col15a1 in cultured human aortic SMC results in a decrease in proliferation and an increase in migration of SMC. Taken together, we hypothesize that SMC-derived COL15A1 plays a critical role in atherosclerosis by promoting matrix organization needed for lesion and fibrous cap stability. Due to ambiguity in identifying dedifferentiated SMC without lineage tracing, the contribution of SMC in producing COL15A1 and how loss of SMC-derived COL15A1 alters plaque morphology and cellular composition is unknown. As such, we generated inducible SMC specific lineage tracing ( Myh11 -CreER T2 ROSA floxed STOP eYFP ApoE -/- ) Col15a1 knockout (SMC- Col15a1 KO ) and wild-type (SMC- Col15a1 WT ) mice and placed them on Western diet for 18 weeks. SMC- Col15a1 KO exhibited a drastic decrease in overall plaque (~4 fold) and vessel (~1.5 fold) size. In addition, SMC- Col15a1 KO have a decrease in overall plaque collagen content (~3 fold) and collagen fiber maturation. With respect to cell type, SMC- Col15a1 KO demonstrated a decrease in overall plaque cell number (DAPI+, ~2 fold) and an increase in the proportion of macrophage (LGALS3+) cells and decrease in the proportion of SMC (YFP+) within plaques. SMC- Col15a1 KO showed a decrease in lesion SMC proliferative capacity in vivo (~2 fold) that was recapitulated in vitro in population doubling proliferation experiments using isolated primary aortic SMC from SMC- Col15a1 WT and SMC- Col15a1 KO . Interestingly, mixing 1:1 of WT and KO SMC lines was able to rescue the proliferation deficit. These studies provide the first evidence that a SMC derived collagen, COL15A1, is a significant and novel player in advanced atherosclerotic plaque progression, stability, and cellular content.

Smooth muscle cells from the anastomosed artery are the major precursors for neointima formation in both artery and vein grafts.

Accumulation of smooth muscle cells (SMC) results in neointima formation in injured vessels. Two graft models consisting of vein and artery grafts were created by anastomosing common carotid arteries to donor vessels. To identify the origin of the neointima cells from anastomosed arteries, we use Wnt1-Cre/reporter mice to label and track SMCs in the common carotid artery. The contribution of SMCs in the neighboring arteries to neointima formation was studied. On evaluating the artery grafts after 1 month, >90 % of the labeled neointima cells were found to have originated from the anastomosing host arteries. Most of the neointima cells were also smooth muscle α-actin positive (SMA-α(+)) and expressed the smooth muscle myosin heavy chain (SMMHC), the SMC terminal differentiation marker. In vein grafts, about 60 % SMA-α-positive cells were from anastomosing arteries. Bone marrow cells did not contribute to neointima SMCs in vein grafts, but did co-stain with markers of inflammatory cells. Wnt1 expression was not detected in the neointima cells in the vein or artery grafts, or the injured femoral arteries. Neointima SMCs showed the synthetic phenotype and were positively labeled with BrdU in vitro and in vivo. Treatment with the IGF-1 receptor inhibitor suppressed SMC proliferation and neointima formation in vein grafts. Our results indicate that SMCs from the neighboring artery are predominantly present in the neointima formed in both vein and artery grafts and that Wnt1-Cre mice can be used to explore the role of SMCs originating from neighboring vessels in vascular remodeling.

Abstract 149: Pluripotency Factors Krüppel-Like Factor 4 and Octamer-binding Transcription Factor 4 Alter Indices of Plaque Stability in Long Term Western Diet Fed Mice by Regulating Smooth Muscle Cell Phenotypes

The bulk of life threatening thrombotic events have been associated with disruption of the fibrous cap, an atheroprotective layer of smooth muscle α-actin positive (ACTA2+) cells that form around the plaque, and the presence of a large foam cell-laden necrotic core within the plaque. Despite the overwhelming research demonstrating that ACTA2+ cells are beneficial for plaque stability, and cells positive for macrophage-markers are detrimental, there are major ambiguities regarding the origins of these cells, and their role in lesion stability. To clearly define the contribution of smooth muscle cells (SMCs) within atherosclerotic lesions, we generated SMC specific lineage tracing Apoe-/- mice containing a SM myosin heavy chain ( Myh11 ) tamoxifen-inducible cre-recombinase gene and a floxed STOP ROSA eYFP gene ( Myh11 YFP ApoE-/- mice) thus allowing activation of eYFP exclusively in fully differentiated SMCs before the onset of atherosclerosis and subsequent determination of the fate of these cells and their progeny irrespective of continued expression of MYH11 or other SMC marker genes. Remarkably, our results reveal that 86% of SMCs cannot be identified using traditional SMC markers, such as ACTA2, and 23% of presumed macrophages (LGALS3+ cells) are derived from SMC origins. The last finding was confirmed in human coronary atheromas using the ISH-PLA approach. SMC specific knockout (KO) of the pluripotency factor Klf4 in Myh11 YFP ApoE-/- mice did not alter the frequency of phenotypically modulated (ACTA2-eYFP+) SMCs within atherosclerotic lesions of mice fed a high fat diet for 18 weeks, however, decreased the number of ACTA2-eYFP+ SMCs that expressed LGALS3, and increased several indices of plaque stability, suggesting a detrimental role for KLF4 in SMCs within atherosclerotic lesions. Conversely, SMC specific Oct4 KO resulted in a dramatic reduction in the number of ACTA2-eYFP+ SMCs within the lesion with marked decreases in indices of plaque stability. In summary results show that the majority of SMC-derived cells within advanced atherosclerotic lesions cannot be identified using conventional SMC marker genes, and that phenotypic switching of SMC during atherogenesis is differentially regulated by the pluripotency factors KLF4 and OCT4.

Age-related increase of stem marker expression influences vascular smooth muscle cell properties

ObjectiveAging represents a major risk factor for vascular disease development. With aging, changes of the biological properties of vascular smooth muscle cells (SMCs) are observed. Stem marker expression characterizes SMCs during developmental growth and atherosclerosis, but the contribution of SMCs with stem features to the age-related arterial remodeling remains largely unknown. Methods and resultsImmunostaining revealed rare vascular growth factor receptor-1+ (flt-1+) and c-kit+ cells in tunica media of grossly normal human young (17–30 years old) large arteries and 2-month old rat aorta, whereas CD133+ cells were absent. In large arteries of human aged donors (64–77 years), flt-1+ and c-kit+ cell number increased in the intimal thickening and tunica media. Double immunofluorescence revealed that 30.6 ± 3% of flt-1+ intimal cells co-expressed α-smooth muscle actin. Immunostaining, blots and RT-PCR documented the increased expression of flt-1 and c-kit in 20–24-month old rat aortic media. In vitro, old rat aortic SMCs proliferated and migrated more with greater flt-1, c-kit, NF-κB, VCAM-1, IAP-1 and MCP-1 levels and less α-smooth muscle actin and myosin compared to young SMCs. Old SMCs were also more susceptible to all-trans retinoic and NF-κB inhibition-induced apoptosis compared to young SMCs. Anti-flt-1 blocking antibody reduced migration and placental growth factor-induced but not serum and PDGF-BB-stimulated proliferation of old SMCs. ConclusionsThe increase of flt-1+ and c-kit+ SMCs characterizes large arteries of aged donors; the blocking of flt-1 signaling influences the behavior of old SMCs, suggesting that the accumulation of SMCs with a stem phenotype contributes to the age-dependent adverse arterial remodeling.

Smooth muscle protein 22 alpha-Cre is expressed in myeloid cells in mice

Background: Experiments using Cre recombinase to study smooth muscle specific functions rely on strict specificity of Cre transgene expression. Therefore, accurate determination of Cre activity is critical to the interpretation of experiments using smooth muscle specific Cre. Methods and results: Two lines of smooth muscle protein 22 α-Cre (SM22α-Cre) mice were bred to floxed mice in order to define Cre transgene expression. Southern blotting demonstrated that SM22α-Cre was expressed not only in tissues abundant of smooth muscle, but also in spleen, which consists largely of immune cells including myeloid and lymphoid cells. PCR detected SM22α-Cre expression in peripheral blood and peritoneal macrophages. Analysis of SM22α-Cre mice crossed with a recombination detector GFP mouse revealed GFP expression, and hence recombination, in circulating neutrophils and monocytes by flow cytometry. Conclusions: SM22α-Cre mediates recombination not only in smooth muscle cells, but also in myeloid cells including neutrophils, monocytes, and macrophages. Given the known contributions of myeloid cells to cardiovascular phenotypes, caution should be taken when interpreting data using SM22α-Cre mice to investigate smooth muscle specific functions. Strategies such as bone marrow transplantation may be necessary when SM22α-Cre is used to differentiate the contribution of smooth muscle cells versus myeloid cells to observed phenotypes.

New therapeutic potential of microRNA treatment to target vulnerable atherosclerotic lesions and plaque rupture

Atherosclerotic lesion vulnerability leading to plaque rupture is a major cause of morbidity in western society. Although several recent major trials have identified statins and angiotensin-converting enzyme inhibitors as having a pleiotropic benefit, no current therapeutic regime directly targets atherosclerosis. The emerging functions of microRNAs (miRs) in regulating gene expression have opened diverse possibilities in understanding plaque biology and in offering new therapeutic strategies. In this review, we consider vascular endothelial cells, smooth muscle cells and monocytes as the main cellular participants in vessel homeostasis during atherosclerosis evolution and discuss how they are functionally modified by miRs and how these modifications may allow therapeutic targeting. Emerging roles for miRs in the pro-inflammatory functions of monocytes and macrophages, and proangiogenic functions of endothelial cells, suggest that miRs regulating these processes are potential targets. Conversely, the contribution of smooth muscle cells to plaque integrity may be augmented by miR-based agents. Recent investigations have uncovered key roles for miRs in each of these areas, which may be targeted through either silencing of proatherogenic or augmentation of antiatherogenic pathways. With emerging miR-based therapeutics, a new paradigm for therapeutic intervention with the ultimate goal of plaque stabilization may exist.

Early Atheroma-Derived Agonists of Peroxisome Proliferator–Activated Receptor-γ Trigger Intramedial Angiogenesis in a Smooth Muscle Cell–Dependent Manner

Neovascularization favors intraplaque hemorrhage and plaque rupture. Development of therapeutic strategies against atheromatous angiogenesis requires elucidation of its initiating factors. We investigated the contribution of smooth muscle cells (SMCs) and atheroma-derived lipids to the initiation of atheroma-associated neoangiogenesis. Forty human aortic segments, each harvested from a different donor, were classified as healthy or as bearing early atheromatous lesions, including fatty streaks and fibrolipidic atheroma, according to their histological features. Immunostaining for blood vessels and vascular endothelial growth factor-A (VEGF-A), as well as measurement of VEGF-A protein and mRNA levels by ELISA and real-time PCR, revealed that angiogenesis and VEGF-A production were enhanced in the medial layer of atheromatous aortas. The intramedial vessel density and invasiveness and the production of VEGF-A by medial SMCs were indeed increased in atheromatous aortas compared with healthy aortas. Furthermore, intimal layers of atheromatous aortas were enriched in soluble lipid mediators capable of inducing a sustained increase in VEGF-A production by medial SMCs, turning these cells into potent inducers of angiogenesis when incorporated into mouse Matrigel implants. Both effects were inhibited by the peroxisome proliferator-activated receptor-γ inhibitor GW9662 and mimicked by its agonist, rosiglitazone. We show that VEGF-A production is upregulated in medial SMCs of human atheromatous aortas and that peroxisome proliferator-activated receptor-γ agonists derived from early intimal lesions are likely to contribute to this phenotypic change. Our findings suggest that medial SMCs are central organizers of an angiogenic response initiated by the subendothelial accumulation of atherogenic lipids.

Smooth Muscle–Specific Deletion of Nitric Oxide–Sensitive Guanylyl Cyclase Is Sufficient to Induce Hypertension in Mice

Arterial hypertension is one of the major diseases in industrial countries and a major cause of mortality. One of the main vascular factors responsible for the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). NO acts predominantly via NO-sensitive guanylyl cyclase (NO-GC), which is made up of 2 different subunits (alpha and beta). Deletion of the beta(1) subunit leads to a global NO-GC knockout, and these mice are hypertensive. However, global deletion of NO-GC in mice does not allow identification of the cell/tissue type responsible for the elevated blood pressure. To determine the relative contribution of smooth muscle cells to the hypertension seen in NO-GC knockout mice, we generated smooth muscle-specific knockout mice for the beta(1) subunit of NO-GC using a tamoxifen-inducible system. Male mice were investigated because the Cre transgene used is located on the Y chromosome. Tamoxifen injection led to a rapid reduction of NO-GC expression in smooth muscle but did not affect that in other tissues. Parallel to a reduction in NO-induced cGMP accumulation, NO-induced relaxation of aortic smooth muscle was gradually lost after induction by tamoxifen. Concomitantly, these animals developed hypertension within 3 to 4 weeks. We generated a model in which the development of hypertension can be visualized over time by deletion of a single gene in smooth muscle cells. In sum, our data provide evidence that deletion of NO-GC solely in smooth muscle is sufficient to cause hypertension.

Protein kinase C delta: a master regulator of apoptosis in neointimal thickening?

Smooth muscle cell (SMC)-driven neointimal hyperplasia is a key mechanism that reduces the long-term benefit of vascular interventions such as stenting, balloon angioplasty, and by-pass grafting. Furthermore, neointimal hyperplasia contributes significantly to the progression of atherosclerotic lesions. In response to arterial injury, neointimal hyperplasia develops rapidly—within a couple of months—as opposed to atherosclerotic lesions, which progress over decades. In general, the contribution of SMC to neointimal thickening can be attributed to increased SMC migration, proliferation, reduced apoptosis, and increased extracellular matrix accumulation. In addition, the recruitment of vascular progenitor cells and constrictive remodelling contribute to lesion formation and luminal narrowing.1,2 All of these mechanisms hold promise for the development of therapeutic interventions, and an impressive body of work has identified numerous potential targets. Unfortunately, many of these interventions were effective in rodent models but failed in human disease. However, in the recent past extensive clinical experience has been collected using drug-eluting stents (DES) for local delivery of antiproliferative drugs. Although highly efficient in inhibiting stent restenosis, sirolimus- and paclitaxel-eluting stents reduce endothelial re-growth and require long-term dual anti-platelet therapy.3 Therefore, new strategies that efficiently inhibit restenosis may still be of clinical … *Corresponding author. Tel: +49 201 723 3460; Fax: +49 201 723 5968. E-mail address : j.fischer{at}uk-essen.de

New Insight in Aetiopathogenesis of Aortic Diseases

Knowledge in the aetiopathogeny of aortic disease helps to characterise aortic lesions better and determine the risk of evolution and therapeutic strategies as well. This article focusses on aneurysms and dissections, and excludes causes related to infection, systemic inflammatory diseases and trauma. The biomedical literature of the past 10 years has been reviewed here. Aortic diseases are heterogeneous along the aorta as far as their genetic determinants, contribution of smooth muscle cells, inflammation and thrombus formation are concerned. Degradation of extracellular matrix by proteases causing aortic disease is a 'terminal' event, modulated by genetic background, haemodynamic strain, cellular events and thrombus formation. New genetic determinants of aortic disease have been identified. Proteases degrading the aortic wall are derived from a variety of cell types in addition to macrophages, including neutrophils on the luminal thrombus, mesenchymal and endothelial cells in the wall. Smooth muscle cells contribute to aortic wall homeostasis against inflammation and proteolysis. The degradation of the wall is followed by, or paralleled with, a failure of aortic reconstruction. Aortic diseases are diverse, and involve a multiplicity of biological systems in the vascular wall and at the interface with blood. Future research needs to unravel distinct cellular and molecular mechanisms causing the clinical events, in particular, dissection, expansion of already formed aneurysms and rupture.

J008 Vascular smooth muscle modulates endothelial control of vasoreactivity via ros production through myoendothelial communications

Endothelial control of vascular smooth muscle plays a major role in the resulting vasoreactivity implicated in physiological or pathological circulatory processes. However, a comprehensive understanding of endothelial (EC) / smooth muscle cells (SMC) crosstalk is far from complete. Here, we have examined the role of gap junctions in this crosstalk and we demonstrate an active contribution of SMC to endothelial control of vasomotor tone. In small intrapulmonary arteries, quantitative RT-PCR, Western Blot analyses revealed the presence of 3 connexins (Cx): 37, 40 and 43. Immunofluorescence labelling showed the presence of Cx 37, 40 and 43 in EC and the presence of Cx 37 and 40 in SMC. Functional experiments showed that the peptides homologous to the gap 26 and 27 domains of the extracellular loops of Cx 43 (43Gap 26) or Cx 37 and 43 (37-43Gap 27) respectively, (1) interrupt intercellular communications in a specific manner, (2) attenuate the calcium and contractile responses to serotonin (5-HT) simultaneously recorded in pulmonary arteries and (3) abolish the diffusion in SMC of carboxyfluorescein-AM loaded in EC. Similarly, contractile and calcium responses to 5-HT were decreased by superoxide dismutase and catalase which, catabolize superoxide anion (O2•-) and H2O2, respectively. Both Cx- and reactive oxygen species (ROS)-mediated effects on the responses to 5-HT were reversed by (1) L-NAME, a NO synthase inhibitor, or (2) endothelium removal with CHAPS. Electron paramagnetic resonance directly demonstrated that 5-HTinduced ROS production, and more specifically O2•-, originated from the SMC. Finally, 5-HT decreased basal cyclic GMP content in isolated intact arteries, whereas a cocktail of 5-HT, superoxide dismutase and catalase increased basal cyclic GMP content. Altogether, the findings suggest that 5-HT produces O2•- in the smooth muscle and NO in the endothelium. O2•- passes through the myoendothelial junctions to decrease endothelial NO production and thus strengthen pulmonary vasoreactivity. This is the first time that we demonstrate a control of the endothelial NO function by the smooth muscle is demonstrated. Since ROS is often overproduced in many cardiovascular diseases, such process could also be of great importance in pathological conditions.

Enhanced spontaneous Ca 2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.