Elastin Deposition Research Articles

Alterations in phenotype and gene expression of adult human aneurysmal smooth muscle cells by exogenous nitric oxide

Abdominal aortic aneurysms (AAA) are characterized by matrix remodeling, elastin degradation, absence of nitric oxide (NO) signaling, and inflammation, influencing smooth muscle cell (SMC) phenotype and gene expression. Little is known about the biomolecular release and intrinsic biomechanics of human AAA-SMCs. NO delivery could be an attractive therapeutic strategy to restore lost functionality of AAA-SMCs by inhibiting inflammation and cell stiffening. We aim to establish the differences in phenotype and gene expression of adult human AAA-SMCs from healthy SMCs. Based on our previous study which showed benefits of optimal NO dosage delivered via S-Nitrosoglutathione (GSNO) to healthy aortic SMCs, we tested whether such benefits would occur in AAA-SMCs. The mRNA expression of three genes involved in matrix degradation (ACE, ADAMTS5 and ADAMTS8) was significantly downregulated in AAA-SMCs. Total protein and glycosaminoglycans synthesis were higher in AAA-SMCs than healthy-SMCs (p < 0.05 for AAA-vs. healthy- SMC cultures) and was enhanced by GSNO and 3D cultures (p < 0.05 for 3D vs. 2D cultures; p < 0.05 for GSNO vs. non-GSNO cases). Elastin gene expression, synthesis and deposition, desmosine crosslinker levels, and lysyl oxidase (LOX) functional activity were lower, while cell proliferation, iNOS, LOX and fibrillin-1 gene expressions were higher in AAA-SMCs (p < 0.05 between respective cases), with differential benefits from GSNO exposure. GSNO and 3D cultures reduced MMPs −2, −9, and increased TIMP-1 release in AAA-SMC cultures (p < 0.05 for GSNO vs. non-GSNO cultures). AAA-SMCs were inherently stiffer and had smoother surface than healthy SMCs (p < 0.01 in both cases), but GSNO reduced stiffness (~25%; p < 0.01) and increased roughness (p < 0.05) of both cell types. In conclusion, exogenously-delivered NO offers an attractive strategy by providing therapeutic benefits to AAA-SMCs.

De novo variants in an extracellular matrix protein coding gene, fibulin-5 (FBLN5) are associated with pseudoexfoliation.

Fibulin-5 (FBLN5), an extracellular scaffold protein, plays a crucial role in the activation of Lysyl oxidase like-1 (LOXL1), a tropoelastin crosslinking enzyme, and subsequent deposition of elastin in the extracellular matrix. Following study identifies polymorphisms within FBLN5 gene as risk factors and its aberrant expression in the pathogenesis of an ocular disorder, pseudoexfoliation (PEX). Exons and exon-intron boundaries within FBLN5 gene were scanned through fluorescence-based capillary electrophoresis for polymorphisms as risk factors for PEX pathogenesis in recruited study subjects with Indian ethnicity. mRNA and protein expression of FBLN5 was checked in lens capsule of study subjects through qRT-PCR and western blotting, respectively. In vitro functional analysis of risk variants was done through luciferase reporter assays. Thirty study subjects from control and PEX affected groups were scanned for potential risk variants. Putative polymorphisms identified by scanning were further evaluated for genetic association in a larger sample size comprising of 338 control and 375 PEX affected subjects. Two noncoding polymorphisms, hg38 chr14:g.91947643G>A (rs7149187:G>A) and hg38 chr14:g.91870431T>C (rs929608:T>C) within FBLN5 gene are found to be significantly associated with PEX as risk factors with a p-value of 0.005 and 0.004, respectively. Molecular assays showed a decreased expression of FBLN5 at both mRNA and protein level in lens capsule of pseudoexfoliation syndrome (PEXS) affected subjects than control. This study unravels two novel risk variants within FBLN5 gene in the pathogenesis of PEX. Further, a decreased expression of FBLN5 in PEXS affected lens capsules implicates a pathogenic link between extracellular matrix maintenance and onset of PEX.

Captopril treatment during development alleviates mechanically induced aortic remodeling in newborn elastin knockout mice.

Deposition of elastin and collagen in the aorta correlates with increases in blood pressure and flow during development, suggesting that the aorta adjusts its mechanical properties in response to hemodynamic stresses. Elastin knockout (Eln-/-) mice have high blood pressure and pathological remodeling of the aorta and die soon after birth. We hypothesized that decreasing blood pressure in Eln-/- mice during development may reduce hemodynamic stresses and alleviate pathological remodeling of the aorta. We treated Eln+/+ and Eln-/- mice with the anti-hypertensive medication captopril throughout embryonic development and then evaluated left ventricular (LV) pressure and aortic remodeling at birth. We found that captopril treatment decreased Eln-/- LV pressure to values near Eln+/+ mice and alleviated the wall thickening and changes in mechanical behavior observed in untreated Eln-/- aorta. The changes in thickness and mechanical behavior in captopril-treated Eln-/- aorta were not due to alterations in measured elastin or collagen amounts, but may have been caused by alterations in smooth muscle cell (SMC) properties. We used a constitutive model to understand how changes in stress contributions of each wall component could explain the observed changes in composite mechanical behavior. Our modeling results show that alterations in the collagen natural configuration and SMC properties in the absence of elastin may explain untreated Eln-/- aortic behavior and that partial rescue of the SMC properties may account for captopril-treated Eln-/- aortic behavior.

Ventricular remodeling of single-chambered myh6-/- adult zebrafish hearts occurs via a hyperplastic response and is accompanied by elastin deposition in the atrium.

Zebrafish (Danio rerio) is widely used as an animal model to understand the pathophysiology of cardiovascular diseases. Here, we present the adult cardiac phenotype of weak atrium, myh6-/-, which carry mutations in the zebrafish atrial myosin heavy chain. Homozygous mutants survive to adulthood and are fertile despite their initial weak atrial beat. In adult mutants, the atrium remains hypoplastic and shows elastin deposition while mutant ventricles exhibit increased size. In mammals, hypertrophy is the most common mechanism resulting in cardiomegaly. Using immunohistochemistry and confocal microscopy to measure cardiomyocyte cell size, density and proliferation, we show that the enlargement of the myh6-/- ventricle is predominantly due to hyperplasia. However, we identified similar transcriptional profiles to the mammalian hypertrophy response via RT-PCR of the hyperplastic ventricles. Furthermore, we show activation of the ER-stress pathway by western blot analysis. In conclusion, we can assume, based on our model, that molecular signaling pathways associated with hypertrophy in mammals, in combination with ER-stress activation, result in hyperplasia in zebrafish. In addition, to our knowledge, this is the first time to report elastin deposition in the atrium.

Dynamics of elastin in liver fibrosis: Accumulates late during progression and degrades slowly in regression.

Elastin is an amorphous protein highly resistant to elastase degradation and is believed to be the most stable component among the extracellular matrix (ECM) members. Thus the excessive deposition of elastin in advanced liver fibrosis may contribute to the declining reversibility of the disease. Our previous study has found that elastin crosslinking inhibition can effectively arrest liver fibrosis progression. To further understand the roles of elastin involved in liver fibrosis, we systematically investigated the expression, accumulation, and degradation based on dynamic and bidirectional CCl4 -induced liver fibrosis mouse models and visualized the ultrastructure of elastin globules in cultured LX-2 cells. We found that the expression pattern of tropoelastin (soluble elastin) and collagen I was not completely comparable at both the transcriptional and posttranscriptional levels during liver fibrosis progression and regression. Elastin mainly accumulated onto the internodular fibrous septa and enlarged portal areas and intertwined with collagen I at the late stage of liver fibrosis. Three-dimensional analysis of elastin and collagen I by confocal immunofluorescence coupled with biochemical analyses revealed that with respect to collagen, elastin deposition was characterized by late aggregation in progression and slow turnover in regression. In addition, we visualized the dynamic ultrastructure of ECM fibers during liver fibrogenesis and fibrolysis and the ultrastructure of elastin globules self-aggregated by tropoelastin crosslinking. Our current study established new general hallmarks of elastin levels and forms in progressive and regressive liver fibrosis and provided a foundation for further experimental investigation of the growing role of elastin in liver fibrosis regression.

Tropoelastin-Coated Tendon Biomimetic Scaffolds Promote Stem Cell Tenogenic Commitment and Deposition of Elastin-Rich Matrix.

Tendon tissue engineering strategies that recreate the biophysical and biochemical native microenvironment have a greater potential to achieve regeneration. Here, we developed tendon biomimetic scaffolds using mechanically competent yarns of poly-ε-caprolactone, chitosan, and cellulose nanocrystals to recreate the inherent tendon hierarchy from a nano-to-macro scale. These were then coated with tropoelastin (TROPO) through polydopamine (PDA) linking, to mimic the native extracellular matrix (ECM) composition and elasticity. Both PDA and TROPO coatings decreased surface stiffness without masking the underlying substrate. We found that human adipose-derived stem cells (hASCs) seeded onto these TROPO biomimetic scaffolds more rapidly acquired their spindle-shape morphology and high aspect ratio characteristic of tenocytes. Immunocytochemistry shows that the PDA and TROPO-coated surfaces boosted differentiation of hASCs toward the tenogenic lineage, with sustained expression of the tendon-related markers scleraxis and tenomodulin up to 21 days of culture. Furthermore, these surfaces enabled the deposition of a tendon-like ECM, supported by the expression of collagens type I and III, tenascin, and decorin. Gene expression analysis revealed a downregulation of osteogenic and fibrosis markers in the presence of TROPO when compared with the control groups, suggesting proper ECM deposition. Remarkably, differentiated cells exposed to TROPO acquired an elastogenic profile due to the evident elastin synthesis and deposition, contributing to the formation of a more mimetic matrix in comparison with the PDA-coated and uncoated conditions. In summary, our biomimetic substrates combining biophysical and biological cues modulate stem cell behavior potentiating their long-term tenogenic commitment and the production of an elastin-rich ECM.

Oxygen Disrupts Human Fetal Lung Mesenchymal Cells. Implications for Bronchopulmonary Dysplasia.

Exogenous mesenchymal stromal cells (MSCs) ameliorate experimental bronchopulmonary dysplasia. Moreover, data from term-born animal models and human tracheal aspirate-derived cells suggest altered mesenchymal signaling in the pathophysiology of neonatal lung disease. We hypothesized that hyperoxia, a factor contributing to the development of bronchopulmonary dysplasia, perturbs human lung-resident MSC function. Mesenchymal cells were isolated from human fetal lung tissue (16-18 wk of gestation), characterized and cultured in conditions resembling either intrauterine (5% O2) or extrauterine (21% and 60% O2) atmospheres. Secretome data were compared with MSCs obtained from term umbilical cord tissues. The human fetal lung mesenchyme almost exclusively contains CD146pos. MSCs expressing SOX-2 and OCT-4, which secrete elastin, fibroblast growth factors 7 and 10, vascular endothelial growth factor, angiogenin, and other lung cell-protecting/-maturing proteins. Exposure to extrauterine atmospheres in vitro leads to excessive proliferation, reduced colony-forming ability, alterations in the cell's surface marker profile, decreased elastin deposition, and impaired secretion of factors important for lung growth. Conversely, umbilical cord-derived MSCs abundantly secreted factors that impaired lung MSCs are unable to produce. Oxygen-impaired human fetal lung MSC function may contribute to disrupted repair capacity and arrested lung growth. Exogenous MSCs may act by triggering the signaling pathways lost by impaired endogenous lung mesenchymal cells.

Exposure to acetaminophen impairs vasodilation, increases oxidative stress and changes arterial morphology of rats.

Acetaminophen (APAP) is one of the most widely consumed drugs in the world. Studies have shown renal and hepatic damage as the direct result of high oxidative stress induced by APAP. Since the cardiovascular system is sensitive to oxidative stress and literature describes increased cardiovascular dysfunction in APAP consumers, this work aimed to evaluate harmful effects of APAP on the vascular system. Rats were exposed to APAP (400mg/kg/day in drinking water) for 14days. Plasma and aortas were collected and stored in - 80°C and a selection of arteries was prepared for isometric tension recordings, morphological, immunohistochemical and protein expression analysis. The APAP-treated group presented increased transaminases (ALT/AST) and malondialdehyde levels in the plasma compared to controls. Lipid peroxidation, glutathione reductase and superoxide dismutase levels were increased in the plasma and arteries of the APAP group. Nevertheless, glutathione level was reduced as compared to control group. The vasodilation response to acetylcholine and sodium nitroprusside (0.1nM to 10µM) was also impaired after APAP treatment; however, the vascular relaxation was restored after treatment with vitamin C (100µM). Arteries from the APAP group presented reduced wall thickness, collagen deposition, elastic fibers and increased immunoreactivity to nitrotyrosine. eNOS and sGC protein expression remained unchanged and were at similar levels as controls. These findings showed higher oxidative stress and impaired vasodilation in rats exposed to APAP. Furthermore, arteries presented reduced cell layers, collagen, elastin deposition and significantly increased immunoreactivity to nitrotyrosine after APAP treatment.

Transforming Growth Factor-β-Neutralizing Antibodies Improve Alveolarization in the Oxygen-Exposed Newborn Mouse Lung.

Abnormal alveolar formation and excessive disordered elastin accumulation are key pathological features in bronchopulmonary dysplasia. Transforming growth factor (TGF)-β is an important regulator of the extracellular matrix in the developing lung. To determine if increased TGF-β would injure alveolar development by activating TGF-β signaling and by influencing the expression of elastogenesis-related protein, we performed intraperitoneal injection of newborn mice with the TGF-β-neutralizing antibody 1D11 and observed whether 1D11 had a protective role in the oxygen (O2)-exposed newborn mouse lung. The newborn mice were exposed to 85% O2 for 14 and 21 days. 1D11 was administered by intraperitoneal injection every day from postnatal days 3 to 20. Alveolar morphology was assessed by hematoxylin and eosin staining. The expression and distribution of elastin were evaluated by immunohistochemistry. The level of TGF-β signaling-related proteins were measured by immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot. The expression levels of elastogenesis-related proteins, including tropoelastin, fibulin-5, and neutrophil elastase (NE), which participate in the synthesis, assembly, and degradation of elastin, were detected by real-time PCR and Western blot. In this research, impaired alveolar development and elastin deposition as well as the excessive activation of TGF-β signaling were observed in the newborn mouse lung exposed to hyperoxia. 1D11 improved alveolarization as well as the distribution of elastin in the newborn lung with hyperoxia exposure. The expression levels of tropoelastin, fibulin-5, and NE, which are important components of elastogenesis, were decreased by treatment with 1D11 in the injured newborn lung. These data demonstrate that 1D11 improved alveolarization by blocking the TGF-β signaling pathway and by reducing the abnormal expression of elastogenesis-related proteins in the O2-exposed newborn mouse lung. 1D11 may become a new therapeutic method to prevent the development of bronchopulmonary dysplasia.

Regeneration of a neoartery through a completely autologous acellular conduit in a minipig model: a pilot study

BackgroundVascular grafts are widely used as a treatment in coronary artery bypass surgery, hemodialysis, peripheral arterial bypass and congenital heart disease. Various types of synthetic and natural materials were experimented to produce tissue engineering vascular grafts. In this study, we investigated in vivo tissue engineering technology in miniature pigs to prepare decellularized autologous extracellular matrix-based grafts that could be used as vascular grafts for small-diameter vascular bypass surgery.MethodsAutologous tissue conduits (3.9 mm in diameter) were fabricated by embedding Teflon tubings in the subcutaneous pocket of female miniature pigs (n = 8, body weight 25–30 kg) for 4 weeks. They were then decellularized by CHAPS decellularization solution. Heparin was covalently-linked to decellularized tissue conduits by Sulfo-NHS/EDC. We implanted these decellularized, completely autologous extracellular matrix-based grafts into the carotid arteries of miniature pigs, then sacrificed the pigs at 1 or 2 months after implantation and evaluated the patency rate and explants histologically.ResultsAfter 1 month, the patency rate was 100% (5/5) while the inner diameter of the grafts was 3.43 ± 0.05 mm (n = 5). After 2 months, the patency rate was 67% (2/3) while the inner diameter of the grafts was 2.32 ± 0.14 mm (n = 3). Histological staining confirmed successful cell infiltration, and collagen and elastin deposition in 2-month samples. A monolayer of endothelial cells was observed along the inner lumen while smooth muscle cells were dominant in the graft wall.ConclusionA completely autologous acellular conduit with excellent performance in mechanical properties can be remodeled into a neoartery in a minipig model. This proof-of-concept study in the large animal model is very encouraging and indicates that this is a highly feasible idea worthy of further study in non-human primates before clinical translation.

Evaluation of cellular ingrowth within porcine extracellular matrix scaffolding in congenital heart disease surgery.

The search for an ideal material for cardiac tissue repair has led to utilization of porcine small intestinal submucosa extracellular matrix (CorMatrix). Here, we examine the histologic features of CorMatrix and the associated cellular growth at a variety of time intervals. Tissues with CorMatrix from ten patients (4 male, 6 female) with ages ranging from 2 weeks to 2 years, and implant duration ranging from 1 week to 2 years were included in this study. Samples for analysis were collected at autopsy. Surgical repair sites included great vessel repair (n=9), atrial septum defect (n=1), coronary vessels (n=1), as well as aortic (n=1) and mitral valve (n=2) leaflets. In all specimens, CorMatrix was composed of dense laminated regions of collagen, without appreciable elastin staining. In most grafts, especially those implanted for extended periods of time, tissue with luminal CD31 positivity covered the intimal surface of the CorMatrix graft. This tissue (neo-intima) consisted of spindled myofibroblasts (SMA) and small CD31 positive vessels with occasional mononuclear cells in a matrix composed of collagen, glycosaminoglycans, and rarely elastin, after extended periods of implantation. These features were readily identified in patients as early as 1 month after CorMatrix implantation. The matrix comprising the CorMatrix itself remained largely acellular, despite implantation times up to 2 years, with degradation of the graft material. We provide a framework for histologic expectations when evaluating explanted CorMatrix grafts. In this regard, the CorMatrix matrix is likely to remain acellular without significant elastin deposition, whereas the intimal and adventitial surfaces become coated by proliferating cells in a novel matrix of collagen and glycosaminoglycans.

Noni Juice and Hydrolyzed Fish Collagen Supplement Improves Skin Elasticity

Morinda citrifolia, more commonly known as noni, is a tropical tree with fruit that has a wide variety of traditional uses and biological effects. It had been used for the maintenance of skin health by Pacific Islanders and has been found to promote collagen deposition in vivo. Oral ingestion of hydrolyzed collagen has also been shown to improve skin quality. A 21-day human intervention study was conducted to evaluate a dietary supplement containing a combination of noni fruit juice and hydrolyzed marine collagen, as well as other ingredients, on skin viscoelastic properties in healthy Japanese adults. Daily ingestion of the supplement significantly improved net elasticity and biological elasticity measurements of the cheek. This also accompanied a trend for lowered skin autofluorescence, a measurement of skin glycation. The results indicate that the combination of noni fruit juice and hydrolyzed collagen improved skin biomechanical properties through enhanced collagen and elastin deposition in the skin, reduced skin glycation and improved skin hydration, possibly from increased hyaluronic acid concentration. Noni fruit juice also effectively scavenged cumene hydroperoxide in vitro. The positive effects on skin quality are likely due, in part, to the antioxidant and antiglycative properties of the supplement, as well as its ability to induce fibroblast proliferation and influence extracellular matrix protein gene expression.

Fibronectin promotes elastin deposition, elasticity and mechanical strength in cellularised collagen-based scaffolds

One of the tightest bottlenecks in vascular tissue engineering (vTE) is the lack of strength and elasticity of engineered vascular wall models caused by limited elastic fiber deposition. In this study, flat and tubular collagen gel-based scaffolds were cellularised with vascular smooth muscle cells (SMCs) and supplemented with human plasma fibronectin (FN), a known master organizer of several extracellular matrix (ECM) fiber systems. The consequences of FN on construct maturation was investigated in terms of geometrical contraction, viscoelastic mechanical properties and deposition of core elastic fiber proteins. FN was retained in the constructs and promoted deposition of elastin by SMCs as well as of several proteins required for elastogenesis such as fibrillin-1, lysyl oxidase, fibulin-4 and latent TGF-β binding protein-4. Notably, gel contraction, tensile equilibrium elastic modulus and elasticity were strongly improved in tubular engineered tissues, approaching the behaviour of native arteries. In conclusion, this study demonstrates that FN exerts pivotal roles in directing SMC-mediated remodeling of scaffolds toward the production of a physiological-like, elastin-containing ECM with excellent mechanical properties. The developed FN-supplemented systems are promising for tissue engineering applications where the generation of mature elastic tissue is desired and represent valuable advanced in vitro models to investigate elastogenesis.

Spatial and temporal changes in extracellular elastin and laminin distribution during lung alveolar development

Lung alveolarization requires precise coordination of cell growth with extracellular matrix (ECM) synthesis and deposition. The role of extracellular matrices in alveogenesis is not fully understood, because prior knowledge is largely extrapolated from two-dimensional structural analysis. Herein, we studied temporospatial changes of two important ECM proteins, laminin and elastin that are tightly associated with alveolar capillary growth and lung elastic recoil respectively, during both mouse and human lung alveolarization. By combining protein immunofluorescence staining with two- and three-dimensional imaging, we found that the laminin network was simplified along with the thinning of septal walls during alveogenesis, and more tightly associated with alveolar endothelial cells in matured lung. In contrast, elastin fibers were initially localized to the saccular openings of nascent alveoli, forming a ring-like structure. Then, throughout alveolar growth, the number of such alveolar mouth ring-like structures increased, while the relative ring size decreased. These rings were interconnected via additional elastin fibers. The apparent patches and dots of elastin at the tips of alveolar septae found in two-dimensional images were cross sections of elastin ring fibers in the three-dimension. Thus, the previous concept that deposition of elastin at alveolar tips drives septal inward growth may potentially be conceptually challenged by our data.

Temporal changes in peritoneal cell phenotype and neoelastic matrix induction with hyaluronan oligomers and TGF-β1 after implantation of engineered conduits.

The neoassembly and maturation of elastic matrix is an important challenge for engineering small-diameter grafts for patients with peripheral artery disease. We have previously shown that hyaluronan oligomers and transforming growth factor-β (elastogenic factors or EFs) promote elastogenesis in smooth muscle cell (SMC) culture. However, their combined effects on macrophages and inflammatory cells in vivo are unknown. This information is needed to use the body (e.g., peritoneal cavity) as an "in vivo bioreactor" to recruit autologous cells to implanted EF-functionalized scaffolds. In this study, we determined if peritoneal fluid cells respond to EFs like smooth muscle cells and if these responses differ between cells sourced during different stages of inflammation triggered by scaffold implantation. Electrospun poly(ε-caprolactone)/collagen conduits were implanted in the peritoneal cavity prior to peritoneal fluid collection at 3-42days postimplantation. Cells from the fluid were cultured in vitro with and without EFs to determine their response. Their phenotype/behaviour was assessed with a DNA assay, quantitative real-time PCR, and immunofluorescence. The EFs reduced peritoneal cell proliferation, maintained cell contractility, and unexpectedly did not exhibit proelastic effects, which we attributed to differences in cell density. We found the greatest elastin deposition in regions containing a high cell density. Further, we found that cells isolated from the peritoneal cavity at longer times after conduit implantation responded better to the EFs and exhibited more CD31 expression than cells at an earlier time point. Overall, this study provides information about the potential use of EFs in vivo and can guide the design of future tissue-engineered vascular grafts.

Experimental Right Ventricular Hypertension Induces Regional β1-Integrin-Mediated Transduction of Hypertrophic and Profibrotic Right and Left Ventricular Signaling.

BackgroundDevelopment of right ventricular (RV) hypertension eventually contributes to RV and left ventricular (LV) myocardial fibrosis and dysfunction. The molecular mechanisms are not fully elucidated.Methods and ResultsPulmonary artery banding was used to induce RV hypertension in rats in vivo. Then, we evaluated cardiac function and regional remodeling 6 weeks after pulmonary artery banding. To further elucidate mechanisms responsible for regional cardiac remodeling, we also mimicked RV hypertensive stress by cyclic mechanical stretching applied to confluent cultures of cardiac fibroblasts, isolated from the RV free wall, septal hinge points, and LV free wall. Echocardiography and catheter evaluation demonstrated that rats in the pulmonary artery banding group developed RV hypertension with leftward septal displacement, LV compression, and increased LV end‐diastolic pressures. Picrosirius red staining indicated that pulmonary artery banding induced marked RV fibrosis and dysfunction, with prominent fibrosis and elastin deposition at the septal hinge points but less LV fibrosis. These changes were associated with proportionally increased expressions of integrin‐β1 and profibrotic signaling proteins, including phosphorylated Smad2/3 and transforming growth factor‐β1. Moreover, mechanically stretched fibroblasts also expressed significantly increased levels of α‐smooth muscle actin, integrin‐β1, transforming growth factor‐β1, collagen I deposition, and wrinkle formation on gel assays, consistent with myofibroblast transformation. These changes were not observed in parallel cultures of mechanically stretched fibroblasts, preincubated with the integrin inhibitor (BTT‐3033).ConclusionsExperimentally induced RV hypertension triggers regional RV, hinge‐point, and LV integrin β1‐dependent mechanotransduction signaling pathways that eventually trigger myocardial fibrosis via transforming growth factor‐β1 signaling. Reduced LV fibrosis and preserved global function, despite geometrical and pressure aberrations, suggest a possible elastin‐mediated protective mechanism at the septal hinge points.

Development of an Off-the-Shelf Tissue-Engineered Sinus Valve for Transcatheter Pulmonary Valve Replacement: a Proof-of-Concept Study.

Tissue-engineered heart valves with self-repair and regeneration properties may overcome the problem of long-term degeneration of currently used artificial prostheses. The aim of this study was the development and in vivo proof-of-concept of next-generation off-the-shelf tissue-engineered sinus valve (TESV) for transcatheter pulmonary valve replacement (TPVR). Transcatheter implantation of off-the-shelf TESVs was performed in a translational sheep model for up to 16weeks. Transapical delivery of TESVs was successful and showed good acute and short-term performance (up to 8weeks), which then worsened over time most likely due to a non-optimized in vitro valve design. Post-mortem analyses confirmed the remodelling potential of the TESVs, with host cell infiltration, polymer degradation, and collagen and elastin deposition. TESVs proved to be suitable as TPVR in a preclinical model, with encouraging short-term performance and remodelling potential. Future studies will enhance the clinical translation of such approach by improving the valve design to ensure long-term functionality.

Pentagalloyl glucose increases elastin deposition, decreases reactive oxygen species and matrix metalloproteinase activity in pulmonary fibroblasts under inflammatory conditions

Emphysema is characterized by degradation of lung alveoli that leads to poor airflow in lungs. Irreversible elastic fiber degradation by matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) activity leads to loss of elasticity and drives the progression of this disease. We investigated if a polyphenol, pentagalloyl glucose (PGG) can increase elastin production in pulmonary fibroblasts. We also studied the effect of PGG treatment in reducing MMP activity and ROS levels in cells. We exposed rat pulmonary fibroblasts to two different types of inflammatory environments i.e., tumor necrosis factor-α (TNF-α) and cigarette smoke extract (CSE) to mimic the disease. Parameters like lysyl oxidase (LOX) and elastin gene expression, MMP-9 activity in the medium, lysyl oxidase (LOX) activity and ROS levels were studied to assess the effect of PGG on pulmonary fibroblasts. CSE inhibited lysyl oxidase (LOX) enzyme activity that resulted in a decreased elastin formation. Similarly, TNF-α treated cells showed less elastin in the cell layers. Both these agents caused increase in MMP activity and ROS levels in cells. However, when supplemented with PGG treatment along with these two inflammatory agents, we saw a significant increase in elastin deposition, reduction in both MMP activity and ROS levels. Thus PGG, which has anti-inflammatory, anti-oxidant properties coupled with its ability to aid in elastic fiber formation, can be a multifunctional drug to potentially arrest the progression of emphysema.

Role of fetal nutrient restriction and postnatal catch-up growth on structural and mechanical alterations of rat aorta.

Intrauterine growth restriction (IUGR), induced by maternal undernutrition, leads to impaired aortic development. This is followed by hypertrophic remodelling associated with accelerated growth during lactation. Fetal nutrient restriction is associated with increased aortic compliance at birth and at weaning, but not in adult animals. This mechanical alteration may be related to a decreased perinatal collagen deposition. Aortic elastin scaffolds purified from young male and female IUGR animals also exhibit increased compliance, only maintained in adult IUGR females. These mechanical alterations may be related to differences in elastin deposition and remodelling. Fetal undernutrition induces similar aortic structural and mechanical alterations in young male and female rats. Our data argue against an early mechanical cause for the sex differences in hypertension development induced by maternal undernutrition. However, the larger compliance of elastin in adult IUGR females may contribute to the maintenance of a normal blood pressure level. Fetal undernutrition programmes hypertension development, males being more susceptible. Deficient fetal elastogenesis and vascular growth is a possible mechanism. We investigated the role of aortic mechanical alterations in a rat model of hypertension programming, evaluating changes at birth, weaning and adulthood. Dams were fed ad libitum (Control) or 50% of control intake during the second half of gestation (maternal undernutrition, MUN). Offspring aged 3days, 21days and 6months were studied. Blood pressure was evaluated in vivo. In the thoracic aorta we assessed gross structure, mechanical properties (intact and purified elastin), collagen and elastin content and internal elastic lamina (IEL) organization. Only adult MUN males developed hypertension (systolic blood pressure: MUNmales =176.6±5.6mmHg; Controlmales =136.1±4.9mmHg). At birth MUN rats were lighter, with smaller aortic cross-sectional area (MUNmales =(1.51±0.08)×105 μm2 , Controlmales =(2.8±0.04)×105 μm2 ); during lactation MUN males and females exhibited catch-up growth and aortic hypertrophy (MUNmales =(14.5±0.5)×105 μm2 , Controlmales =(10.4±0.9)×105 μm2 ), maintained until adulthood. MUN aortas were more compliant until weaning (functional stiffness: MUNmales =1.0±0.04; Controlmales =1.3±0.03), containing less collagen with larger IEL fenestrae, returning to normal in adulthood. Purified elastin from young MUN offspring was more compliant in both sexes; only MUN adult females maintained larger elastin compliance (slope: MUNfemales =24.1±1.9; Controlfemales =33.3±2.8). Fetal undernutrition induces deficient aortic development followed by hypertrophic remodelling and larger aortic compliance in the perinatal period, with similar alterations in collagen and elastin in both sexes. The observed alterations argue against an initial mechanical cause for sex differences in hypertension development. However, the maintenance of high elastin compliance in adult females might protect them against blood pressure rise.

Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

Pulmonary fibrosis is a progressive scarring disorder of the lung with dismal prognosis and no curative therapy. Clusterin, an extracellular chaperone and regulator of cell functions, is reduced in bronchoalveolar lavage fluid of patients with pulmonary fibrosis. However, its distribution and role in normal and fibrotic human lung are incompletely characterized. Immunohistochemical localization of clusterin revealed strong staining associated with fibroblasts in control lung and morphologically normal areas of fibrotic lung but weak or undetectable staining in fibrotic regions and particularly fibroblastic foci. Clusterin also co-localized with elastin in vessel walls and additionally with amorphous elastin deposits in fibrotic lung. Analysis of primary lung fibroblast isolates in vitro confirmed the down-regulation of clusterin expression in fibrotic compared with control lung fibroblasts and further demonstrated that TGF-β1 is capable of down-regulating fibroblast clusterin expression. shRNA-mediated down-regulation of clusterin did not affect TGF-β1-induced fibroblast-myofibroblast differentiation but inhibited fibroblast proliferative responses and sensitized to apoptosis. Down-regulation of clusterin in fibrotic lung fibroblasts at least partly due to increased TGF-β1 may therefore represent an appropriate but insufficient response to limit fibroproliferation. Reduced expression of clusterin in the lung may also limit its extracellular chaperoning activity contributing to dysregulated deposition of extracellular matrix proteins.