Extracellular Matrix Fibrosis Research Articles

Immunohistochemical Expression of the SERPINA3 Protein in Uterine Fibroids.

SERPINA3 (α-1-antichymotrypsin, AACT, ACT) is produced by the liver and released into plasma in an anti-inflammatory response and plays a role as a modulator of extracellular matrix (ECM) by inhibiting serine proteases. Numerous studies proved an increased level of SERPINA3 in many types of cancer, which could be linked to SERPINA3's anti-apoptotic function. In the context of progressive ECM fibrosis during the development of uterine fibroids, which are one of the most common hypertrophic changes within the uterus, it is interesting to describe the level of SERPINA3 protein in this type of lesion and the surrounding tissues. We used immunohistochemical staining of the SERPINA3 protein and compared the intensity of the signal between the myoma tissue and the surrounding normal tissue. We showed a surprising reduction in the amount of the SERPINA3 protein within uterine fibroids compared to surrounding tissues. This observation sheds new light on the role of this protein in the formation of proliferative changes and suggests that understanding the mechanism of its action may become the basis for the development of new diagnostic and therapeutic tools.

Inactivation of ERK1/2 in cancer-associated hepatic stellate cells suppresses cancer-stromal interaction by regulating extracellular matrix in fibrosis.

The ERK1/2 pathway is involved in epithelial-mesenchymal transformation and cell cycle of tumor cells in hepatocellular carcinoma (HCC). In the present study, we investigated the involvement of ERK1/2 activation on hepatic stellate cells (HSCs). We identified ERK1/2 phosphorylation in activated HSCs of HCC samples. We found that tumor cells promoted the migration and invasion capacity of HSCs by activating ERK1/2 phosphorylation. Using high throughput transcriptome sequencing analysis, we found that ERK1/2 inhibition altered genes significantly correlated to signaling pathways involved in extracellular matrix remodeling. We screened genes and demonstrated that the ERK1/2 inhibition-related gene set significantly correlated to cancer-associated fibroblast infiltration in TCGA HCC tumor samples. Moreover, inhibition of ERK1/2 suppressed tumor cell-induced enhancement of HSC migration and invasion by regulating expression of fibrosis markers FAP, FN1 and COL1A1. In a tumor cell and HSC splenic co-transplanted xenograft mouse model, inhibition of ERK1/2 suppressed liver tumor formation by downregulating fibrosis, indicating ERK1/2 inhibition suppresses tumor-stromal interactions in vivo. Taken together, our data indicate that inhibition of ERK1/2 in tumor-associated HSCs suppresses tumor-stromal interactions and progression. Furthermore, inhibition of ERK1/2 may be a potential target for HCC treatment.

Therapeutic In Situ Cancer Vaccine Using Pulsed Stereotactic Body Radiotherapy-A Translational Model.

Both radiation and cancer therapeutic vaccine research are more than 100 years old, and their potential is likely underexplored. Antiangiogenics, nanoparticle targeting, and immune modulators are some other established anticancer therapies. In the meantime, immunotherapy usage is gaining momentum in clinical applications. This article proposes the concept of a pulsed/intermittent/cyclical endothelial-sparing single-dose in situ vaccination (ISVRT) schedule distinguishable from the standard therapeutic stereotactic body radiotherapy (SBRT) and stereotactic radiosurgery (SRS) plans. This ISVRT schedule can repeatedly generate tumor-specific neoantigens and epitopes for primary and immune modulation effects, augment supplementary immune enhancement techniques, activate long-term memory cells, avoid extracellular matrix fibrosis, and essentially synchronize with the vascular normalized immunity cycle. The core mechanisms of ISVRT impacting in situ vaccination would be optimizing cascading antigenicity and adjuvanticity. The present proposed hypothesis can be validated using the algorithm presented. The indications for the proposed concept are locally progressing/metastatic cancers that have failed standard therapies. Immunotherapy/targeted therapy, chemotherapy, antiangiogenics, and vascular-lymphatic normalization are integral to such an approach.

Principles for diagnosing heart failure with preserved ejection fraction

Low-grade systemic inflammation, myocardial stress, and extracellular matrix fibrosis lead to heart failure with preserved ejection fraction (HFpEF). The HFA-PEFF diagnostic algorithm and the H2FPEF score are recommended for detecting HFpEF. Their low compliance is the reason for improving the methods for diagnosing HFpEF. Modern paraclinical diagnostics of HFpEF includes an assessment of the left ventricular filling pressure during diastolic stress test. Phase analysis of left atrial strain during resting echocardiography may be promising to conclude an increase in mean left atrial pressure. Research interest is growing in relation to biomarkers involved in the regulation of collagen synthesis. Together, paraclinical diagnostics help to characterize sequential morphofunctional cardiac remodeling, increasing the possibility of HFpEF detection.

Expression analysis of MMP14: Key enzyme action in modulating visceral adipose tissue plasticity in patients with obesity.

Compromised adipose tissue plasticity is a hallmark finding of obesity orchestrated by the intricate interplay between various extracellular matrix components. Collagen6 (COL6) is well characterized in obese visceral adipose tissue (VAT), not much is known about MMP14 which is hypothesized to be the key player in matrix reorganization. Subjects with obesity (BMI ≥40; n = 50) aged 18-60 years undergoing bariatric surgery and their age-matched controls (BMI < 25; n = 30) were included. MMP14, Col6A3 and Tissue inhibitor of metalloproteinase 2 (TIMP2) mRNA expression was assessed in VAT and their serum levels along with endotrophin were estimated in both groups preoperatively and post-operatively in the obese group. The results were analysed statistically and correlated with anthropometric and glycaemic parameters, namely fasting glucose and insulin, HbA1c, HOMA-IR, HOMA-β and QUICKI. Circulating levels as well as mRNA expression profiling revealed significant differences between the individuals with and without obesity (p < .05), more so in individuals with diabetes and obesity (p < .05). Follow-up serum analysis revealed significantly raised MMP14 (p < .001), with decreased Col6A3, endotrophin and TIMP2 levels (p < .01, p < .001 and p < .01, respectively). A rise in serum MMP14 protein, simultaneous with post-surgical weight loss and decreased serum levels of associated extracellular matrix (ECM) remodellers, suggests its crucial role in modulating obesity-associated ECM fibrosis and pliability of VAT.

Collagen type I alters the proteomic signature of macrophages in a collagen morphology-dependent manner

Idiopathic pulmonary fibrosis is a progressive lung disease that causes scarring and loss of lung function. Macrophages play a key role in fibrosis, but their responses to altered morphological and mechanical properties of the extracellular matrix in fibrosis is relatively unexplored. Our previous work showed functional changes in murine fetal liver-derived alveolar macrophages on fibrous or globular collagen morphologies. In this study, we applied differential proteomics to further investigate molecular mechanisms underlying the observed functional changes. Macrophages cultured on uncoated, fibrous, or globular collagen-coated plastic were analyzed by liquid chromatography-mass spectrometry. The presence of collagen affected expression of 77 proteins, while 142 were differentially expressed between macrophages grown on fibrous or globular collagen. Biological process and pathway enrichment analysis revealed that culturing on any type of collagen induced higher expression of enzymes involved in glycolysis. However, this did not lead to a higher rate of glycolysis, probably because of a concomitant decrease in activity of these enzymes. Our data suggest that macrophages sense collagen morphologies and can respond with changes in expression and activity of metabolism-related proteins. These findings suggest intimate interactions between macrophages and their surroundings that may be important in repair or fibrosis of lung tissue.

Role of Epithelial-to-Mesenchymal Transition of Retinal Pigment Epithelial Cells in Glaucoma Cupping

Optic nerve head (ONH) cupping is a clinical feature of glaucoma associated with extracellular matrix (ECM) remodelling and lamina cribrosa (LC) fibrosis. Peripapillary atrophy (PPA) occurs commonly in glaucoma, and is characterised by the loss of retinal pigment epithelium (RPE) adjacent to the ONH. Under pro-fibrotic conditions, epithelial cells throughout the body can differentiate into fibroblast-like cells through epithelial-to-mesenchymal transition (EMT) and contribute to ECM fibrosis. This is investigated here in the context of glaucoma and PPA. Human-donor ONH sections were assessed for the presence of the RPE cell-specific marker RPE65 using immunofluorescence. We examined the EMT response of ARPE-19 cells to the following glaucoma-related stimuli: cyclic mechanical stretch, mechanical stiffness, transforming growth factor beta (TGFβ), and tumour necrosis factor alpha (TNFα). The gene expression was measured using the PCR of the epithelial tight junction marker zona occludens 1 (ZO-1) and the mesenchymal markers alpha smooth muscle actin (αSMA) and vimentin. A scratch assay was used to assess the ARPE-19 migration. Significant RPE-65 staining was demonstrated in the glaucomatous ONH. The cyclic stretching and substrate stiffness of the ARPE-19 cells caused a significant decrease in ZO-1 (p = 0.04), and an increase in αSMA (p = 0.04). The scratch assays demonstrated increased migration of ARPE19 in the presence of TNFα (p = 0.02). Furthermore, ARPE-19 cells undergo an EMT-like transition (gain of αSMA, loss of ZO-1 and increased migration) in response to glaucomatous stimuli. This suggests that during PPA, RPE cells have the potential to migrate into the ONH and differentiate into fibroblast-like cells, contributing to glaucomatous ONH cupping.

Does Myocardial Atrophy Represent Anti-Arrhythmic Phenotype?

This review focuses on cardiac atrophy resulting from mechanical or metabolic unloading due to various conditions, describing some mechanisms and discussing possible strategies or interventions to prevent, attenuate or reverse myocardial atrophy. An improved awareness of these conditions and an increased focus on the identification of mechanisms and therapeutic targets may facilitate the development of the effective treatment or reversion for cardiac atrophy. It appears that a decrement in the left ventricular mass itself may be the central component in cardiac deconditioning, which avoids the occurrence of life-threatening arrhythmias. The depressed myocardial contractility of atrophied myocardium along with the upregulation of electrical coupling protein, connexin43, the maintenance of its topology, and enhanced PKCƐ signalling may be involved in the anti-arrhythmic phenotype. Meanwhile, persistent myocardial atrophy accompanied by oxidative stress and inflammation, as well as extracellular matrix fibrosis, may lead to severe cardiac dysfunction, and heart failure. Data in the literature suggest that the prevention of heart failure via the attenuation or reversion of myocardial atrophy is possible, although this requires further research.

Rhein ameliorates transverse aortic constriction-induced cardiac hypertrophy via regulating STAT3 and p38 MAPK signaling pathways.

The progression from compensatory hypertrophy to heart failure is difficult to reverse, in part due to extracellular matrix fibrosis and continuous activation of abnormal signaling pathways. Although the anthraquinone rhein has been examined for its many biological properties, it is not clear whether it has therapeutic value in the treatment of cardiac hypertrophy and heart failure. In this study, we report for the first time that rhein can ameliorate transverse aortic constriction (TAC)-induced cardiac hypertrophy and other cardiac damage in vivo and in vitro. In addition, rhein can reduce cardiac hypertrophy by attenuating atrial natriuretic peptide, brain natriuretic peptide, and β-MHC expression; cardiac fibrosis; and ERK phosphorylation and transport into the nucleus. Furthermore, the inhibitory effect of rhein on myocardial hypertrophy was similar to that of specific inhibitors of STAT3 and ERK signaling. In addition, rhein at therapeutic doses had no significant adverse effects or toxicity on liver and kidney function. We conclude that rhein reduces TAC-induced cardiac hypertrophy via targeted inhibition of the molecular function of ERK and downregulates STAT3 and p38 MAPK signaling. Therefore, rhein might be a novel and effective agent for treating cardiac hypertrophy and other cardiovascular diseases.

P144 a Transforming Growth Factor Beta Inhibitor Peptide, Generates Antifibrogenic Effects in a Radiotherapy Induced Fibrosis Model.

Radiation-induced fibrosis (RIF) is a severe side effect related with soft tissues sarcomas (STS) radiotherapy. RIF is a multicellular process initiated primarily by TGF-β1 that is increased in irradiated tissue, whose signaling leads to intracellular Smad2/3 phosphorylation and further induction of profibrotic target genes. P144 (Disetertide©) is a peptide inhibitor of TGF-β1 and is proposed as a candidate compound for reducing RIF associated wound healing problems and muscle fibrosis in STS. Methods: A treatment and control group of WNZ rabbits were employed to implement a brachytherapy animal model, through catheter implantation at the lower limb. Two days after implantation, animals received 20 Gy isodosis, intended to induce a high RIF grade. The treatment group received intravenous P144 administration following a brachytherapy session, repeated at 24–72 h post-radiation, while the control group received placebo. Four weeks later, affected muscular tissues underwent histological processing for collagen quantification and P-Smad2/3 immunohistochemistry through image analysis. Results: High isodosis Brachytherapy produced remarkable fibrosis in this experimental model. Results showed retained macro and microscopical morphology of muscle in the P144 treated group, with reduced extracellular matrix fibrosis, with a lower area of collagen deposition measured through Masson’s trichrome staining. Intravenous P144 also induced a significant reduction in Smad2/3 phosphorylation levels compared with the placebo group. Conclusions: P144 administration clearly reduces RIF and opens a new potential co-treatment approach to reduce complications in soft tissue sarcoma (STS) radiotherapy. Further studies are required to establish whether the dosage and timing optimization of P144 administration, in different RIF phases, might entirely avoid fibrosis associated with STS brachytherapy.

Prognostic value of microRNAs in heart failure: A meta-analysis.

Background:Reported studies have shown that expression levels of microRNAs (miRNAs) are related to survival time of patients with heart failure (HF). A systematic review and meta-analysis were conducted to study circulating miRNAs expression and patient outcome.Methods:Meta-analysis estimating expression levels of circulating miRNAs in HF patients from January 2010 until June 30, 2018, through conducting online searches in Pub Med, Cochrane Database of Systematic, EMBASE and Web of Science and reviewed by 2 independent researchers. Using pooled hazard ratio with a 95% confidence interval to assess the correlation between miRNAs expression levels and overall survival.Results:Four relevant articles assessing 19 circulating miRNAs in 867 patients were included. In conclusion, the meta-analysis results suggest that HF patients with low expression of serum miR-1, miR-423-5p, miR-126, miR-21, miR-23, miR-30d, miR-18a-5p, miR-16-5p, miR-18b-5p, miR-27a-3p, miR-26b-5p, miR-30e-5p, miR-106a-5p, miR-233-3P, miR-301a-3p, miR-423-3P, and miR-128 have significantly worse overall survival (P < .05). Among them, miR-18a-5p, miR-18b-5p, miR-30d, miR-30e-5p, and miR-423-5p are strong biomarkers of prognosis in HF.

A Novel Stable Isotope Approach Demonstrates Surprising Degree of Age-Related Decline in Skeletal Muscle Collagen Proteostasis.

Age-related deterioration in turnover of collagen proteins accelerates extracellular matrix fibrosis and hinders adaptation to external stimuli. This project sought to understand factors that increase skeletal muscle fibrosis with age by studying what we term the dynamic protein pool. We hypothesized that the dynamic protein pool size of muscle collagen decreases with age, thus indicating a decrease in proteostatic maintenance (ie, ability to maintain proteostasis), and that failure to account for these changes impacts the interpretation of tracer-measured synthesis rates. We used deuterium oxide (D2O) labeling for up to 60 days in adult (6 months) and old (23 months) mice. The dynamic protein pool in adult skeletal muscle was 65% in tibialis anterior (TA), but only 28% in gastrocnemius (Gastroc). In aged muscle, the dynamic protein pool was further decreased to only 35% and 14% for TA and Gastroc, respectively. We showed that this loss in dynamic pool size was associated with increases in markers of fibrosis and decreased proteostatic maintenance. We demonstrate that aged muscle has higher rates of collagen protein synthesis and lower rates of collagen protein breakdown, which causes collagen accumulation. We further demonstrated that the normal assumption of complete protein renewal and the standard practice of taking a single sample with isotope labeling have profound impacts on interpretation of the genesis of fibrosis. Strategies to maintain muscle function with aging should focus on the dynamic protein pool with attention to methodological strategies to assess those changes.

Relationships between circulating galectin-3, extracellular matrix fibrosis and outcomes in dilated cardiomyopathy.

Galectin-3 is an emerging biomarker in cardiovascular disease. Myocardial galectin-3 is involved in the pathology of cardiac fibrosis; however, the role of circulating galectin-3 is not yet established. To assess the relationships between circulating galectin-3, fibrosis and outcomes in dilated cardiomyopathy (DCM). We included 70 patients (age: 48 ±12.1 years, ejection fraction (EF) 24.4 ±7.4%) with new-onset DCM (n = 35, ≤6 months). Galectin-3 and procollagen type I and III (PICP, PINP, PIIICP, and PIIINP), transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), osteopontin (OPN), matrix metalloproteinases (MMP-2 and -9), and tissue inhibitor (TIMP-1) were determined in serum at baseline and after 3 and 12 months. Patients underwent endomyocardial biopsy. The endpoint was a combination of death and urgent hospitalization at 12 months. Galectin-3 did not correlate with biopsy-determined fibrosis. Baseline galectin-3 correlated with OPN,, TIMP-1, PIIICP, and MMP-2. In new-onset DCM, galectin-3 levels at baseline were higher than at 3 and 12 months, whereas in chronic DCM there was no difference. Galectin-3 was a predictor of the endpoint (hazard ratio (HR) = 1.115; 95% confidence interval (95% CI) = 1.009-1.231; p < 0.05). The best cut-off value was 14.54 ng/mL (area under the curve (AUC) = 0.67). Patients with galectin-3 ≥14.54 ng/mL had an increased risk of events (HR = 2.569; 95% CI = 1.098-6.009; p < 0.05). Circulating galectin-3 is unrelated to fibrosis. Serial measurements of galectin-3 correlated with markers of fibrosis, including markers of collagen synthesis and OPN. Circulating galectin-3 was independently associated with cardiovascular (CV) outcomes in DCM.

Kinetics of selected serum markers of fibrosis in patients with dilated cardiomyopathy and different grades of diastolic dysfunction of the left ventricle.

Fibrosis of the extracellular matrix (ECM) in dilated cardiomyopathy (DCM) is common and compromises both systolic and diastolic function. The aim of this study was to investigate the kinetics of ECM fibrosis markers over a 12 month follow-up in patients with DCM based on the severity of diastolic dysfunction (DD). Seventy consecutive DCM patients (48 ± 12.1 years, ejection fraction 24.4 ± 7.4%) were included in the study. The grade of DD was determined using the ASE/EACVI algorithm. Markers of ECM fibrosis were measured at baseline and at 3 and 12 month follow-ups: collagen type I and III (PICP, PINP, PIIICP, PIIINP), transforming growth factor beta-1 (TGF1-b), connective tissue growth factor (CTGF) and galectin-3 were measured. Patients were divided into three groups according to DD severity: 30 patients with grade I, 18 with grade II and 22 with grade III of DD. Levels of PICP, PINP were increased over a 12-month period, while PIIINP decreased and PIIICP unchanged. Levels of TGF1-b decreased from the 3 to the 12-month points in grade I and II DD, and in grade III they remained unchanged. Levels of CTGF decreased over 12 months in grade III DD but were unchanged in grades I and II. Galectin-3 levels remained the same over all observation periods, irrespective of DD grade. Regardless of the DD grade, markers of collagen type I synthesis increased, markers of collagen type III decreased. Levels of TGF and CTGF had a tendency to decrease. Galectin-3 was revealed not to be a marker discriminating the severity of DD.

Abstract 17177: Furosemide Promotes Myocardial Fibrosis in Swine Heart Failure

Introduction: Although frequently used in heart failure (HF), long-term furosemide exacerbates HF in a swine model. This paradoxical phenomenon may reflect furosemide-induced alterations in signaling proteins of the extracellular matrix (ECM) to increase fibrosis. Examination of the ECM may clarify how treatments like furosemide enhance the progression of HF. Hypothesis: The administration of furosemide increased inflammation and fibrosis of the heart, leading to accelerated HF deterioration in a swine model. Methods: After Institutional Animal Care and Use Committee (IACUC) approval, Yorkshire swine (N=10, 5 = furosemide, 5 = saline) were paced 3 to 5 weeks at 200 beats per minute to induce HF (left ventricular fractional shortening &lt;16% on echocardiogram). Animals were treated with furosemide (1 mg/kg intramuscularly) or saline. Western Blot and histology with Masson’s trichrome stain were performed on transmural LV myocardium to quantify critical determinants of fibrosis. ANOVA with Tukey HSD correction and descriptive statistics were performed using SPSS with significance by α of 0.05; mean ± SEM. Results: The overall increase in ECM fibrosis in HF was clearly demonstrated on Masson’s trichrome histology associated with significant, uniform increase in signaling pathways leading to fibrosis associated with furosemide use. SMAD 2-HF Furosemide to Control (p&lt;.05): HF Furosemide 0.266 ±.05, HF Saline 0.183±.02, Control 0.163±.01 ERK-all (p&lt;.05): HF Furosemide 0.312±.02, HF Saline 0.209±.01,Control 0.149±.01 GDF-15-HF Furosemide to Control (p&lt;.05): HF Furosemide 0.095 ±.00, HF Saline 0.073±.01, Control 0.062±.01 MMP-14-all (p&lt;.05): HF Furosemide 0.451±.02, HF Saline 0.374±.03,Control 0.231±.02 TIMP-1-all (p&lt;.05): HF Furosemide 0.184±.01, HF Saline 0.0135±.03,Control 0.033±.01 PAI-1-all (p&lt;.05): HF Furosemide 0.105±.01, HF Saline 0.046±.01,Control 0.043±.00 Conclusions: Histologic and biochemical analysis showed worsening HF in furosemide-treated paced swine was associated with consistent increases in fibrosis and indices of adverse ECM remodeling. The diuretic intended to reduce water retention during HF appeared to enhance myocardial fibrosis, paradoxically accelerating pathological processes responsible for HF.

Histopathological changes in the anterior segment with anterior and posterior chamber intraocular lens

ObjectivePatients have shown a lowering of intraocular pressure (IOP) after cataract surgery. Histopathology studies have reported trabecular meshwork (TM) changes in pseudophakic eyes with posterior chamber intraocular lens (PCIOL) and have eluded to the mechanisms for IOP decrease. Unlike PCIOLs, TM histopathology changes after implantation of an anterior chamber intraocular lens (ACIOL) have not been studied, to our knowledge. Therefore, this study aims to examine the histopathological changes in both the TM and corneal endothelium among donor eyes with ACIOL, PCIOL, and phakic eyes. MethodsForty fixed postmortem donor eyes were obtained, sectioned, and embedded. Slides were stained with Masson's trichrome and CD31 vascular endothelial antibody, and further digitalized. Customized Medical Parachute TMAN software quantified the cellular components, the trabecular extracellular matrix (ECM), ECM fibrosis, and trabecular area. Schlemm's canal and corneal endothelium were quantified across the ACIOL, PCIOL, and phakic groups. ResultsCellular area component of the TM was lower in the ACIOLs and PCIOLs than in phakic eyes, but statistically significant only between PCIOL and phakic eyes (p = 0.0023). ECM area component, TM fibrosis score and TM lamellae area, ciliary process fibrosis, and CD31 expression in Schlemm's canal showed no differences (p = 0.40, 0.99, 0.10, 0.83, 0.45). Significantly lower corneal endothelial cells were seen in ACIOLs compared with both PCIOLs and phakic eyes (p = 0.0002). ConclusionsACIOLs and PCIOLs in our sample group showed that there is loss of cellular components in the TM compared with the phakic eyes, with PCIOLs displaying the least amount of TM cells statistically, in this cohort. The ACIOLs led to a greater loss of corneal endothelial cells than both PCIOLs and phakic eyes after cataract surgery. The endothelial cells in Schlemm's canal did not seem to be affected by the IOL placements. Therefore, this study illustrates that there are histopathological differences seen with the placements of ACIOLs in both TM and cornea.

Tachycardia‐Induced Matrix Metalloproteinases Activation Associated with Collagen Type III Cardiac Fibrosis and Heart Failure in Swine

Cardiovascular disease remains the most common cause of death in the developed world, with heart failure (HF) increasing in prevalence. In the HF disease state, the left ventricle (LV) manifests a robust plasticity response called remodeling. Cardiac remodeling is a dynamic process that can lead to myocardial fibrosis, which is associated with an increased risk of sudden cardiac death. The extracellular matrix (ECM) is a dynamic support structure that is remodeled following cardiac injury and HF. All of the ECM constituents are vulnerable to the diverse stresses on the heart. Within the ECM, matrix metalloproteinases (MMPs), a key family of proteolytic enzymes, alter the interactions between different structural and metabolically active interstitial molecules and play a major role in the structure and function of the ECM.After obtaining Institutional Animal Care and Use Committee (IACUC) approval, Yorkshire swine (N=10) were paced for 3 to 5 weeks with implanted devices at 200 beats per minute. Prior to termination, all 10 paced swine had left ventricular shortening fraction &lt;16%. Ten additional healthy swine served as controls.The results demonstrated differences between control and HF animals consistent with the HF state. Early biomarkers of HF extracellular‐signal regulated kinase 1 and 2 (ERK1/2) (32%), galectin‐3 (25%), Membrane Type 1 MMP (MMP‐14) (68%) and Tissue Inhibitor of Metallo‐Proteinase‐1 (TIMP‐1) (45%) were elevated (by the percentages shown) in the HF animals (p≤0.05) compared to controls. Oxidative stress may have a key role in the transition from compensated hypertrophy to heart failure. In this study, elevated expression of key transcription factors NFκ‐B p65 (39%) and NRF‐2 (62%) (p≤0.05) was seen in HF animals, which is consistent with elevated ROS and inflammation in the myocardium, thus supporting the idea that oxidative stress may have an adverse effect on the heart.Fluorescent staining of left ventricular myocardial tissue sections demonstrated an increase in all collagens of HF animals relative to controls. The overall increase in ECM fibrosis in HF was clearly demonstrated on Masson’s trichrome histology, including the changes in the ratios of structural collagens in the heart. The control tissue shows a small percentage of collagen, whereas HF tissue shows visible increases in Types I, III and VI collagens. Furthermore, the HF tissue also demonstrated a shifting of the Type I/Type III ratio, with Type III, a more elastic collagen, becoming more preponderant and thus allowing for the structural and functional changes consistent with typical HF. Additional exploration into the mechanisms of cardiac remodeling, may lead to treatments to prevent adverse ECM changes and slow the progression of adverse remodeling and ultimately reduce the risk of progression to lethal HF.Support or Funding InformationUniformed Services University Early Career Scientist Award, USU Grant # R089354417

In-Vivo Study of Passive Musculotendon Mechanics in Chronic Hemispheric Stroke Survivors

We characterized the passive mechanical properties of the affected and contralateral musculotendon units in 9 chronic stroke survivors as well as in 6 neurologically-intact controls. Using a position-controlled motor, we precisely indented the distal tendon of the biceps brachii to a 20 mm depth from skin, recording both its sagittal motion using ultrasound movies and the compression force at the tip of the indenter. Length changes of 8 equally-spaced features along the aponeurosis axis were quantified using a pixel-tracking protocol. We report that, on the aggregate and with respect to contralateral and control, respectively, the affected side initiates feature motion at a shorter indentation distance by 61% and 50%, travels further by 15% and 9%, at a lower rate of 28% and 15%, and is stiffer by 40% and 57%. In an extended analysis including the spatial location of the 8 designated features, we report that in contrast to the contralateral and control muscles, the affected musculotendon unit does not strain measurably within the imaging window. These results confirm that chronic stroke-induced spasticity changes musculotendon unit passive mechanics, causing it to not strain under stretch. The mechanisms responsible for altered passive mechanics may lie within extracellular matrix fibrosis.

Twelve-month kinetics of circulating fibrosis-linked microRNAs (miR-21, miR-29, miR-30, and miR-133a) and the relationship with extracellular matrix fibrosis in dilated cardiomyopathy.

IntroductionA single measurement of any biomarker may not reflect its full biological meaning. The kinetics of fibrosis-linked microRNAs and their relationship with extracellular matrix (ECM) fibrosis in dilated cardiomyopathy (DCM) have not been explored.Material and methodsWe evaluated 70 consecutive DCM patients (48 ±12.1 years, left ventricular ejection fraction 24.4 ±7.4%). All patients underwent right ventricular endomyocardial biopsy in order to quantify ECM fibrosis and measure collagen volume fraction (CVF). Circulating microRNAs (miR-21-5p, miR-29b, miR-30c-5p, and miR-133a-3p) were measured with quantitative polymerase chain reaction (PCR) at baseline and at 3 and 12 months.ResultsBased on the biopsy results, two groups of patients were identified: with (n = 24, 34.3%) and without (n = 46, 65.7%) ECM fibrosis. Except for a single measurement of miR-29b at 3 months (DCM with fibrosis: 6.03 ±0.72 vs. DCM without fibrosis: 6.4 ±0.75 ΔCq; p < 0.05), baseline, 3- and 12-month kinetics of microRNAs did not differ between the two groups. Moreover, 12-month microRNA kinetics did not differ in patients with new-onset DCM (duration < 6 months; n = 35) and chronic DCM (> 6 months; n = 35). Only miR-29 at 3 months correlated with CVF (r = –0.31; p < 0.05), whereas other microRNAs did not correlate with CVF either at 3 or at 12 months.ConclusionsRegardless of ECM fibrosis status or duration of the disease, 12-month patterns of circulating microRNAs are similar in DCM. Correlations between microRNAs, measured at 3 and 12 months, are lower than expected. In this study, regardless of the time point, circulating microRNAs were not able to differentiate between DCM patients with versus without fibrosis.

Effect of Concomitant Chronic Obstructive Pulmonary Disease on Markers of Collagen Metabolism in Patients with Ischemic Heart Disease

To study parameters of collagen myocardial metabolism in patients with ischemic cardiomyopathy (ICMP) and to assess the effect of concomitant chronic obstructive pulmonary disease (COPD) on their levels. We examined 130 patients with ICMP (mean age 55.5 [43; 63] years) and 42 patients with ICMP and COPD (mean age 54.8 [41; 63] years). Determination of serum levels of matrix metalloproteinase-1 (MMP-1), its tissue inhibitor (TIMP-1), transforming growth factor-β1 (TGF-β1), and autoantibodies to collagen types I and III was carried out with the help of commercial test systems based on the method of solidphase enzyme immunoassay. Calculation of the volume fraction of interstitial collagen (VFIC) of the myocardium was carried out by the method of J. Shirani. In patients with ICMP combined with COPD, as well as in patients with ICMP we found disbalance in the metabolism of collagen of the extracellular matrix. At the same time, in patients with ICMP+COPD, processes of collagen degradation at the background of extracellular matrix fibrosis were found to be even more pronounced than in patients with ICMP. This was evidenced by the absence of statistically significant differences in the myocardial VFIC (p=0.703), and the level of TGF-β1 (p=0.074), as well as statistically significant higher activity of MMP-1 (p=0.037), MMP-1 / TIMP-1 ratio (p=0.045), and production of autoantibodies to collagen types I and III (p=0.042, p=0.039, respectively). The presence of COPD in patients with ICMP is associated with increased disbalance of the proteolysis-antiproteolysis system in the direction of collagen breakdown that can lead to more pronounced structural changes of the myocardium, and, as consequence, to aggravate functional impairment of the organ.