Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-ß.

Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2(-/-) mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-ß). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-ß activation and Acta2(-/-) SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-ß, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutations.

HANS SELYE: AN INSPIRING TEACHER.

The present writing is a recollection of Hans Selye, as an educator of graduate and post-graduate students. His main aim in teaching his students was to encourage originality and significance of all scientific research.

Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.

The discovery of the myofibroblast has opened new perspectives for the comprehension of the biological mechanisms involved in wound healing and fibrotic diseases. In recent years, many advances have been made in understanding important aspects of myofibroblast basic biological characteristics. This review summarizes such advances in several fields, such as the following: i) force production by the myofibroblast and mechanisms of connective tissue remodeling; ii) factors controlling the expression of α-smooth muscle actin, the most used marker of myofibroblastic phenotype and, more important, involved in force generation by the myofibroblast; and iii) factors affecting genesis of the myofibroblast and its differentiation from precursor cells, in particular epigenetic factors, such as DNA methylation, microRNAs, and histone modification. We also review the origin and the specific features of the myofibroblast in diverse fibrotic lesions, such as systemic sclerosis; kidney, liver, and lung fibrosis; and the stromal reaction to certain epithelial tumors. Finally, we summarize the emerging strategies for influencing myofibroblast behavior in vitro and in vivo, with the ultimate goal of an effective therapeutic approach for myofibroblast-dependent diseases.

Epithelial-mesenchymal crosstalk alteration in kidney fibrosis.

The incidence of chronic kidney diseases (CKD) is constantly rising, reaching epidemic proportions in the western world and leading to an enormous threat, even to modern health-care systems, in industrialized countries. Therapies of CKD have greatly improved following the introduction of drugs targeting the renin-angiotensin system (RAAS) but even this refined pharmacological approach has failed to stop progression to end-stage renal disease (ESRD) in many individuals. In vitro historical data and recent new findings have suggested that progression of renal fibrosis might occur as a result of an altered tubulo-interstitial microenvironment and, more specifically, as a result of an altered epithelial-mesenchymal crosstalk. Here we the review biological findings that support the hypothesis of an altered cellular crosstalk in an injured local tubulo-interstitial microenvironment leading to renal disease progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Myofibroblast-mediated adventitial remodeling: an underestimated player in arterial pathology.

The arterial adventitia has been long considered an essentially supportive tissue; however, more and more data suggest that it plays a major role in the modulation of the vascular tone by complex interactions with structures located within intima and media. The purpose of this review is to summarize these data and to describe the mechanisms involved in adventitia/media and adventitia/intima cross-talk. In response to a plethora of stimuli, the adventitia undergoes remodeling processes, resulting in positive (adaptive) remodeling, negative (constrictive) remodeling, or both. The differentiation of the adventitial fibroblast into myofibroblast (MF), a key player of wound healing and fibrosis development, is a hallmark of negative remodeling; this can lead to vessel stenosis and thus contribute to major cardiovascular diseases. The mechanisms of fibroblast-to-MF differentiation and the role of the MF in adventitial remodeling are highlighted herein.

Transient expression of cellular retinol-binding protein-1 during cardiac repair after myocardial infarction.

Retinoic acid (RA) is a vitamin A derivative that exerts pleiotropic biological effects. Intracellular transport and metabolism of RA are regulated by cellular retinol-binding proteins (CRBP). CRBP-1 is transiently expressed in granulation tissue fibroblasts during wound healing; however, its role in cardiac remodeling remains unknown. A rat myocardial infarction (MI) model was established by ligation of the left coronary artery, and hearts were obtained at 3, 6, 15, 30 and 45 days after operation. Heart sections were examined immunohistochemically using anti-vimentin, anti-α-smooth muscle actin (α-SMA), anti-matrix metalloproteinase (MMP)-2, anti-MMP-9 and anti-CRBP-1 antibodies. Infarction involved 48.8 ± 3.6% of the left ventricle and was followed by an important cardiac remodeling. Vimentin-positive fibroblastic cells including α-SMA-positive myofibroblasts expressed CRBP-1 at 3-, 6-, and 15-days after MI. Expression of CRBP-1 reached a maximum at 6-days after infarction. Thereafter, CRBP-1 expression was dramatically decreased, showing a similar tendency to MMP expression. Human heart specimens of individuals with a recent myocardial infarction demonstrated presence of CRBP-1-positive fibroblasts by immunohistochemistry. We have demonstrated that CRBP-1 is transiently expressed by fibroblasts during cardiac remodeling. Our results suggest that CRBP-1 plays a role in ventricular remodeling after MI allegedly through its RA binding activity.

Autologous transplantation of culture-born myofibroblasts into intact and injured rabbit ligaments.

PURPOSE: The myofibroblast, a contractile fibroblastic cell expressing α-smooth muscle actin (α-SMA), has been reported to play a role in ligament healing. The aim of this study was to evaluate the feasibility of transplanting culture-derived myofibroblasts in injured rabbit medial collateral ligaments (MCL) and in intact anterior cruciate ligaments (ACL). METHODS: Fibroblasts isolated from the iliotibial band were cultured in the presence of transforming growth factor beta-1 (TGF-ß1) for five days and analysed for α-SMA expression. In a concentration of TGF-ß1 ≥ 10 ng/ml, the differentiation rate into myofibroblast was 90%. After labelling with PKH26, α-SMA -positive cells were transplanted in intact ACL and in injured MCL of ten rabbits. RESULTS: Survival of PKH-26+ cells was seen in all intact and damaged ligaments one day after injection. The density of PKH-26+ cells had decreased at seven days postinjection in both ligaments. Double-positive PKH-26+/α-SMA+ cells were only observed in injured MCL at seven days postinjection. Moreover, we found that genetically modified fibroblasts differentiate into myofibroblasts and can be transplanted into ligaments. CONCLUSIONS: Our data demonstrate that culture-born myofibroblasts survive and maintain α-SMA expression up to one week after transplantation. This study provides the first insight into the feasibility of transplanted mechanically active cells for ligament reconstruction.

Drug-eluting stent: importance of clinico-pathological correlations.

The benefit of drug-eluting stents (DES) is the remarkable reduction in the rates of both restenosis and target lesion revascularization. However, the risk of thrombotic complications extends further in DES-implanted arteries compared with those treated with bare-metal stents (BMS). Moreover, in-stent thrombosis (IST) and delayed arterial healing in DES-treated arteries have been identified by histological examination. At autopsy, proliferation of a monolayer composed of endothelium-like cells over stent struts in DES receiving arteries has been observed; however, these cells are negative for well-accepted endothelial cell markers. An inflammatory reaction against the stent struts is apparent after implantation of BMS and paclitaxel-eluting stents, whereas after sirolimus-eluting stents (SES), minimal inflammation is seen up to 6 months after device implantation. IST and in-stent restenosis, both possibly related to a hypersensitivity phenomena, are peculiar to DES, albeit relatively infrequent. A case of enhanced neointimal hyperplasia at 6 months after SES implantation with massive inflammatory reaction including eosinophils, and fibrin deposition is reported here. Observation of the morphological alterations after DES implantation by imaging techniques may furnish important information, but lack of precise comparative data between vascular imaging and histopathology leads to improper interpretation of imaging. Ex vivo imaging using angioscopy, intravascular ultrasound, and optical coherency tomography of SES implantation is presented and the images are compared with the corresponding pathological section.

Regulation of myofibroblast activities: calcium pulls some strings behind the scene.

Myofibroblast-induced remodeling of collagenous extracellular matrix is a key component of our body's strategy to rapidly and efficiently repair damaged tissues; thus myofibroblast activity is considered crucial in assuring the mechanical integrity of vital organs and tissues after injury. Typical examples of beneficial myofibroblast activities are scarring after myocardial infarct and repair of damaged connective tissues including dermis, tendon, bone, and cartilage. However, deregulation of myofibroblast contraction causes the tissue deformities that characterize hypertrophic scars as well as organ fibrosis that ultimately leads to heart, lung, liver and kidney failure. The phenotypic features of the myofibroblast, within a spectrum going from the fibroblast to the smooth muscle cell, raise the question as to whether it regulates contraction in a fibroblast- or muscle-like fashion. In this review, we attempt to elucidate this point with a particular focus on the role of calcium signaling. We suggest that calcium plays a central role in myofibroblast biological activity not only in regulating contraction but also in mediating intracellular and extracellular mechanical signals, structurally organizing the contractile actin-myosin cytoskeleton, and establishing lines of intercellular communication.

50 Years of Myofibroblasts: How the Myofibroblast Concept Evolved.

Myofibroblasts are key cells in mediating normal wound contraction and promoting connective tissue deformations characteristic of fibrosis and scarring. Five decades ago, myofibroblasts were discovered in electron micrographs of wound granulation tissue as fibroblastic cells containing microfilaments that are organized in bundles like those present in smooth muscle. The contractile function of myofibroblasts was demonstrated by measuring the contraction of strips of granulation tissue in response to smooth muscle agonists and in cell culture. Although formation of contractile bundles already defines the myofibroblast, neo-expression of α-smooth muscle actin (α-SMA) in fibroblastic cells has become the most widely used myofibroblast marker. Because α-SMA incorporation into stress fibers mediates enhanced fibroblast contraction, it has been proposed and successfully tested as a drug target in therapeutic approaches to reduce tissue contractures. Other anti-fibrosis strategies target growth factor-, extracellular matrix-, and mechanical stress-induced pathways of myofibroblast activation from various precursors or aim to induce myofibroblast apoptosis. To understand the involved mechanisms of myofibroblast formation and function, critical experimental tools and animal models have been developed, which are made available in this collection of protocols by experts in the field.

Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells.

Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis.

Alpha actin isoforms expression in human and rat adult cardiac conduction system.

In the adult heart, cardiac muscle comprises the working myocardium and the conduction system (CS). The latter includes the sinoatrial node (SAN), the internodal tract or bundle (IB), the atrioventricular node (AVN), the atrioventricular bundle (AVB), the bundle branches (BB) and the peripheral Purkinje fibers (PF). Most of the information concerning the phenotypic features of CS tissue derives from the characterization of avian and rodent developing hearts; data concerning the expression of actin isoforms in adult CS cardiomyocytes are scarce. Using specific antibodies, we investigated the distribution of alpha-skeletal (alpha-SKA), alpha-cardiac (alpha-CA), alpha-smooth muscle (alpha-SMA) actin isoforms and other muscle-typical proteins in the CS of human and rat hearts at different ages. SAN and IB cardiomyocytes were characterized by the presence of alpha-SMA, alpha-CA, calponin and caldesmon, whereas alpha-SKA and vimentin were absent. Double immunofluorescence demonstrated the co-localisation of alpha-SMA and alpha-CA in I-bands of SAN cardiomyocytes. AVN, AVB, BB and PF cardiomyocytes were alpha-SMA, calponin, caldesmon and vimentin negative, and alpha-CA and alpha-SKA positive. No substantial differences in actin isoform distribution were observed in human and rat hearts, except for the presence of isolated subendocardial alpha-SMA positive cardiomyocytes co-expressing alpha-CA in the ventricular septum of the rat. Aging did not influence CS cardiomyocyte actin isoform expression profile. These findings support the concept that cardiomyocytes of SAN retain the phenotype of a developing myogenic cell throughout the entire life span.

The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo.

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of alpha-smooth muscle actin (alpha-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of alpha-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of alpha-skeletal actin has no effect on myofibroblasts, whereas the NH2-terminal peptide of beta-cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing alpha-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of alpha-SMA in wound contraction. The alpha-SMA-FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.

Intimal smooth muscle cells of porcine and human coronary artery express S100A4, a marker of the rhomboid phenotype in vitro.

We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S- and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with alpha-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S- and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein. In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes.

Fascia Is Able to Actively Contract and May Thereby Influence Musculoskeletal Dynamics: A Histochemical and Mechanographic Investigation.

Fascial tissues form a ubiquitous network throughout the whole body, which is usually regarded as a passive contributor to biomechanical behavior. We aimed to answer the question, whether fascia may possess the capacity for cellular contraction which, in turn, could play an active role in musculoskeletal mechanics. Human and rat fascial specimens from different body sites were investigated for the presence of myofibroblasts using immunohistochemical staining for α-smooth muscle actin (n = 31 donors, n = 20 animals). In addition, mechanographic force registrations were performed on isolated rat fascial tissues (n = 8 to n = 18), which had been exposed to pharmacological stimulants. The density of myofibroblasts was increased in the human lumbar fascia in comparison to fasciae from the two other regions examined in this study: fascia lata and plantar fascia [H(2) = 14.0, p < 0.01]. Mechanographic force measurements revealed contractions in response to stimulation by fetal bovine serum, the thromboxane A2 analog U46619, TGF-ß1, and mepyramine, while challenge by botulinum toxin type C3-used as a Rho kinase inhibitor- provoked relaxation (p < 0.05). In contrast, fascial tissues were insensitive to angiotensin II and caffeine (p < 0.05). A positive correlation between myofibroblast density and contractile response was found (r s = 0.83, p < 0.001). The hypothetical application of the registered forces to human lumbar tissues predicts a potential impact below the threshold for mechanical spinal stability but strong enough to possibly alter motoneuronal coordination in the lumbar region. It is concluded that tension of myofascial tissue is actively regulated by myofibroblasts with the potential to impact active musculoskeletal dynamics.

Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome.

BACKGROUND: The endoplasmic reticulum (ER) responds to various stresses by upregulation of ER chaperones, but prolonged ER stress eventually causes apoptosis. Although apoptosis is considered to be essential for the progression and rupture of atherosclerotic plaques, the influence of ER stress and apoptosis on rupture of unstable coronary plaques remains unclear. METHODS AND RESULTS: Coronary artery segments were obtained at autopsy from 71 patients, and atherectomy specimens were obtained from 40 patients. Smooth muscle cells and macrophages in the fibrous caps of thin-cap atheroma and ruptured plaques, but not in the fibrous caps of thick-cap atheroma and fibrous plaques, showed a marked increase of ER chaperone expression and apoptotic cells. ER chaperones also showed higher expression in atherectomy specimens from patients with unstable angina pectoris than in specimens from those with stable angina. Expression of 7-ketocholesterol was increased in the fibrous caps of thin-cap atheroma compared with thick-cap atheroma. Treatment of cultured coronary artery smooth muscle cells or THP-1 cells with 7-ketocholesterol induced upregulation of ER chaperones and apoptosis, whereas these changes were prevented by antioxidants. We also investigated possible signaling pathways for ER-initiated apoptosis and found that the CHOP (a transcription factor induced by ER stress)-dependent pathway was activated in unstable plaques. In addition, knockdown of CHOP expression by small interfering RNA decreased ER stress-dependent death of cultured coronary artery smooth muscle cells and THP-1 cells. CONCLUSIONS: Increased ER stress occurs in unstable plaques. Our findings suggest that ER stress-induced apoptosis of smooth muscle cells and macrophages may contribute to plaque vulnerability.

Sudden coronary death in the young: Evidence of contractile phenotype of smooth muscle cells in the culprit atherosclerotic plaque.

BACKGROUND: Culprit coronary atherosclerotic plaques (APs) from young sudden cardiac death (SCD) victims are mostly non-atheromatous, i.e., consisting of proliferative smooth muscle cells (SMCs). Coronary vasospasm has been advocated to explain plaque instability in the absence of thrombosis. Our aim was to characterize the SMC phenotype in the intima and media of coronary arteries from young SCD victims. METHODS AND RESULTS: A total of 38 coronary artery segments were studied: (a) 18 APs from young (≤40 years old) SCD patients, (b) 9 APs from old (>40 years old) SCD patients, (c) 11 non-atherosclerotic coronary arteries from young patients (≤40 years old). Markers of differentiated SMCs such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chains (SMMHCs), and heavy-caldesmon (h-CaD), were assessed in intima and media by immunohistochemistry and quantified morphometrically. In the intima, their expression was higher in non-atherosclerotic arteries (44.37 ±â€¯3.03% for α-SMA, 14.21 ±â€¯2.01% for SMMHCs, 8.90 ±â€¯1.33% for h-CaD) and APs from young SCD victims (38.95 ±â€¯2.29% for α-SMA, 11.92 ±â€¯1.92% for SMMHCs, 8.93 ±â€¯1.12% for h-CaD) compared with old patients (22.01 ±â€¯3.56% for α-SMA, 6.39 ±â€¯0.7% for SMMHCs, 3.00 ±â€¯0.57% for h-CaD; all P statistically significant). The media of non-atherosclerotic arteries and APs from young SCD victims exhibited strong positivity for the differentiation markers unlike that of old patients. CONCLUSIONS: SMCs of coronary APs as well as from the underlying media from young SCD victims exhibit strong contractile phenotype. In the setting of critical stenosis, both intima and media SMC contractility might contribute to transient coronary spasm leading to myocardial ischemia and SCD.

Cytostatic drugs differentially affect phenotypic features of porcine coronary artery smooth muscle cell populations.

We studied the effects of cytostatic drugs on porcine coronary artery spindle-shaped (S) and rhomboid (R) smooth muscle cell (SMC) biological activities related to intimal thickening (IT) formation. Imatinib, and to a lesser extent curcumin, decreased proliferation of S- and R-SMCs and migratory and urokinase activities of R-SMCs more efficiently compared with cyclosporine plus rapamycin. Imatinib increased the expression of alpha-smooth muscle actin in both SMC populations and that of smoothelin in S-SMCs. It decreased S100A4 expression in R-SMCs. By promoting SMC quiescence and differentiation imatinib and curcumin may represent valid candidates for restenosis preventive and therapeutic strategies.