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    Role of melatonin in sleep deprivation-induced intestinal barrier dysfunction in mice. Gao Ting,Wang Zixu,Dong Yulan,Cao Jing,Lin Rutao,Wang Xintong,Yu Zhengquan,Chen Yaoxing Journal of pineal research Intestinal diseases caused by sleep deprivation (SD) are severe public health threats worldwide. This study focuses on the effect of melatonin on intestinal mucosal injury and microbiota dysbiosis in sleep-deprived mice. Mice subjected to SD had significantly elevated norepinephrine levels and decreased melatonin content in plasma. Consistent with the decrease in melatonin levels, we observed a decrease of antioxidant ability, down-regulation of anti-inflammatory cytokines and up-regulation of pro-inflammatory cytokines in sleep-deprived mice, which resulted in colonic mucosal injury, including a reduced number of goblet cells, proliferating cell nuclear antigen-positive cells, expression of MUC2 and tight junction proteins and elevated expression of ATG5, Beclin1, p-P65 and p-IκB. High-throughput pyrosequencing of 16S rRNA demonstrated that the diversity and richness of the colonic microbiota were decreased in sleep-deprived mice, especially in probiotics, including Akkermansia, Bacteroides and Faecalibacterium. However, the pathogen Aeromonas was markedly increased. By contrast, supplementation with 20 and 40 mg/kg melatonin reversed these SD-induced changes and improved the mucosal injury and dysbiosis of the microbiota in the colon. Our results suggest that the effect of SD on intestinal barrier dysfunction might be an outcome of melatonin suppression rather than a loss of sleep per se. SD-induced intestinal barrier dysfunction involved the suppression of melatonin production and activation of the NF-κB pathway by oxidative stress. 10.1111/jpi.12574
    The Interstitial System of the Brain in Health and Disease. Shetty Ashok K,Zanirati Gabriele Aging and disease The brain interstitial fluid (ISF) and the cerebrospinal fluid (CSF) cushion and support the brain cells. The ISF occupies the brain interstitial system (ISS), whereas the CSF fills the brain ventricles and the subarachnoid space. The brain ISS is an asymmetrical, tortuous, and exceptionally confined space between neural cells and the brain microvasculature. Recently, with a newly developed measuring technique, a series of discoveries have been made in the brain ISS and the drainage of ISF. The goal of this review is to confer recent advances in our understanding of the brain ISS, including its structure, function, and the various processes mediating or disrupting ISF drainage in physiological and pathological conditions. The brain ISF in the deep brain regions has recently been demonstrated to drain in a compartmentalized ISS instead of a highly connected system, together with the drainage of ISF into the cerebrospinal fluid (CSF) at the surface of the cerebral cortex and the transportation from CSF into cervical lymph nodes. Besides, accumulation of tau in the brain ISS in conditions such as Alzheimer's disease and its link to the sleep-wake cycle and sleep deprivation, clearance of ISF in a deep sleep via increased CSF flow, novel approaches to remove beta-amyloid from the brain ISS, and obstruction to the ISF drainage in neurological conditions are deliberated. Moreover, the role of ISS in the passage of extracellular vesicles (EVs) released from neural cells and the rapid targeting of therapeutic EVs into neural cells in the entire brain following an intranasal administration, and the promise and limitations of ISS based drug delivery approaches are discussed. 10.14336/AD.2020.0103
    Effect of cognitive load and emotional valence of distractors on performance during sleep extension and subsequent sleep deprivation. Alger Sara E,Brager Allison J,Balkin Thomas J,Capaldi Vincent F,Simonelli Guido Sleep STUDY OBJECTIVES:The purpose of the present study was to assess the extent to which sleep extension followed by sleep deprivation impacts performance on an attentional task with varying cognitive and attentional demands that influence decisions. METHODS:Task performance was assessed at baseline, after 1 week of sleep extension, and after 40 h of total sleep deprivation. RESULTS:One week of sleep extension resulted in improved performance, particularly for high cognitive load decisions regardless of the emotional salience of attentional distractors. Those who extended sleep the most relative to their habitual sleep duration showed the greatest improvement in general performance during sleep extension. However, a higher percentage of time spent in slow-wave sleep (SWS) on the last night of the sleep extension phase was negatively correlated with performance on more difficult high cognitive load items, possibly reflecting a relatively higher level of residual sleep need. Sleep deprivation generally resulted in impaired performance, with a nonsignificant trend toward greater performance decrements in the presence of emotionally salient distractors. Performance overall, but specifically for high cognitive load decisions, during total sleep deprivation was negatively correlated with longer sleep and higher SWS percentage during subsequent recovery sleep. CONCLUSIONS:The present findings suggest two possibilities: those who performed relatively poorly during sleep deprivation were more vulnerable because (1) they utilized mental resources (i.e. accrued sleep debt) at a relatively faster rate during wakefulness, and/or (2) they failed to "pay down" pre-study sleep debt to the same extent as better-performing participants during the preceding sleep extension phase. 10.1093/sleep/zsaa013
    Extracellular Matrix in Vascular Disease, Part 2/4: JACC Focus Seminar. Barallobre-Barreiro Javier,Loeys Bart,Mayr Manuel,Rienks Marieke,Verstraeten Aline,Kovacic Jason C Journal of the American College of Cardiology Medium-sized and large arteries consist of 3 layers: the tunica intima, tunica media, and tunica adventitia. The tunica media accounts for the bulk of the vessel wall and is the chief determinant of mechanical compliance. It is primarily composed of circumferentially arranged layers of vascular smooth muscle cells that are separated by concentrically arranged elastic lamellae; a form of extracellular matrix (ECM). The tunica media is separated from the tunica intima and tunica adventitia, the innermost and outermost layers, respectively, by the internal and external elastic laminae. This second part of a 4-part JACC Focus Seminar discusses the contributions of the ECM to vascular homeostasis and pathology. Advances in genetics and proteomics approaches have fostered significant progress in our understanding of vascular ECM. This review highlights the important role of the ECM in vascular disease and the prospect of translating these discoveries into clinical disease biomarkers and potential future therapies. 10.1016/j.jacc.2020.03.018
    Napping and weekend catchup sleep do not fully compensate for high rates of sleep debt and short sleep at a population level (in a representative nationwide sample of 12,637 adults). Leger Damien,Richard Jean-Baptiste,Collin Olivier,Sauvet Fabien,Faraut Brice Sleep medicine INTRODUCTION:Short total sleep time (TST < 6 h) is a strong major health determinant that correlates with numerous metabolic, cardiovascular and mental comorbidities, as well as accidents. Our aim was to better understand, at a population level, how adults adapt their TST during the week, and how short sleepers and those with sleep debt and sleep restriction use napping or catching up on sleep during weekends (ie, sleep debt compensation by sleeping longer), which may prevent these comorbidities. METHODS:A large representative sample of 12,367 subjects (18-75 years old) responded by phone to questions about sleep on a national recurrent health poll (Health Barometer, Santé Publique France 2017) assessing sleep schedules (TST) at night, when napping, and over the course of a 24-h period while using a sleep log on workdays and weekends. Retained items were: (1) short sleep (TST ≤ 6 h/24 h); (2) chronic insomnia (international classification of sleep disorders third edition, ICSD-3 criteria); (3) sleep debt (self-reported ideal TST - TST > 60 min, severe > 90 min); and (4) sleep restriction (weekend TST - workday TST = 1-2 h, severe > 2 h). RESULTS:Average TST/24 h was 6h42 (± 3 min) on weekdays and 7h26 (± 3 min) during weekends. In addition, 35.9% (± 1.0%) of the subjects were short sleepers, 27.7% (± 1.0%) had sleep debt (18.8% (± 0.9%) severe), and 17.4% (± 0.9%) showed sleep restriction (14.4% (± 0.8%) severe). Moreover, 27.4% (± 0.9%) napped at least once per week on weekdays (average: 8.3 min (± 0.5 min)) and 32.2% (± 1.0%) on weekend days (13.7 min (± 0.7 min)). Of the 24.2% (± 0.9%) of subjects with severe sleep debt (> 90 min), only 18.2% (± 1.6%) balanced their sleep debt by catching up on sleep on weekends (14.9% (± 0.8%) of men and 21.5% (± 0.9%) of women), and 7.4% (± 1.2%) of these subjects balanced their sleep debt by napping (7.8% (± 0.5%) of men and 6.6% (± 0.4%) of women). The remaining 75.8% (± 5.4%) did not do anything to balance their severe sleep debt during the week. DISCUSSION AND CONCLUSIONS:Short sleep, sleep debt, and sleep restriction during weekdays affected about one third of adults in our study group. Napping and weekend catch-up sleep only compensated for severe sleep debt in one in four subjects. 10.1016/j.sleep.2020.05.030
    Facilitating reproducible research through direct connection of data analysis with manuscript preparation: StatTag for connecting statistical software to Microsoft Word. Welty Leah J,Rasmussen Luke V,Baldridge Abigail S,Whitley Eric W JAMIA open Objectives:To enhance reproducible research by creating a broadly accessible, free, open-source software tool for connecting Microsoft Word to statistical programs (R/R Markdown, Python, SAS, Stata) so that results may be automatically updated in a manuscript. Materials and Methods:We developed StatTag for Windows as a Microsoft Word plug-in using C# and for macOS as a native application using Objective-C. Source code is available under the MIT license at https://github.com/stattag. Results:StatTag links analysis file(s) (R/R Markdown, SAS, Stata, or Python) and a Word document, invokes the statistical program(s) to obtain results, and embeds selected output in the document. StatTag can accommodate multiple statistical programs with a single document and features an interface to view, edit, and rerun statistical code directly from Word. Discussion and Conclusion:StatTag may facilitate reproducibility within increasingly multidisciplinary research teams, improve research transparency through review and publication, and complement data-sharing initiatives. 10.1093/jamiaopen/ooaa043
    The value of lactate/albumin ratio for predicting the clinical outcomes of critically ill patients with heart failure. Guo Wenqin,Zhao Lingyue,Zhao Hanjun,Zeng Fanfang,Peng Changnong,Guo Wenyu,Yan Hongbing Annals of translational medicine Background:Previous studies have shown that the lactate/albumin (L/A) ratio plays a role in predicting the outcomes of septic shock or severe sepsis. However, the role of the L/A ratio in predicting the outcomes of critically ill patients with heart failure remains unclear. We therefore performed a retrospective study to clarify this issue. Methods:The study was based on the Multiparameter Intelligent Monitoring in Intensive Care III (MIMIC-III) database and included critically ill adult patients with heart failure. The primary endpoints were 28-day and 1-year all-cause mortality after admission at the intensive care unit. Results:We analyzed 4,562 patients in this study. We divided the participants into five groups according to the L/A ratio: quintile (Q)1 (L/A ratio ≤0.40, n=913), Q2 (0.40< L/A ratio ≤0.51, n=912), Q3 (0.51< L/A ratio ≤0.66, n=912), Q4 (0.66< L/A ratio ≤0.92, n=912), and Q5 (L/A ratio >0.92, n=913). After stratifying by L/A ratio, the risk of 28-day and 1-year mortality were significantly different between the groups (log-rank P<0.001). Compared with the first quintile, the second, third, fourth, and fifth quintiles of the L/A ratio were associated with higher 28-day [hazard ratio (HR) 1.57, 95% confidence interval (CI): 1.21-2.03 for Q3, HR 1.72, 95% CI: 1.34-2.21 for Q4, and HR 3.15, 95% CI: 2.47-4.01 for Q5) and 1-year mortality (HR 1.19, 95% CI: 1.00-1.41 for Q2, HR 1.36, 95% CI: 1.15-1.60 for Q3, HR 1.42, 95% CI: 1.20-1.67 for Q4, and HR 2.46, 95% CI: 2.09-2.89 for Q5). The restricted cubic spline showed that the L/A ratio positively correlated with both 28-day and 1-year all-cause mortality. Conclusions:The L/A ratio could serve as a predictor of short and long-term mortality in critically ill patients with heart failure. 10.21037/atm-20-4519
    Correction to Lancet Respir Med 2021; published online Jan 28. https://doi.org/10.1016/S2213-2600(20)30517-8. The Lancet. Respiratory medicine 10.1016/S2213-2600(21)00093-X
    Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways. Sun Peibei,Wang Yayan,Gao Tian,Li Kun,Zheng Dongwang,Liu Ajuan,Ni Ya Reproductive biology and endocrinology : RB&E BACKGROUND:Heat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s). As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37. Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility. Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways. METHODS:Human sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation. Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation. The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining. The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments. Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation. RESULTS:Human sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation. In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex. Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased. Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38. Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation. CONCLUSIONS:Taken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction. In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways. 10.1186/s12958-021-00723-2
    [Congenital Pulmonary Artery Stenoses as a Rare Cause of Pulmonary Hypertension]. Kovacs Z,Guth S,Fistera D,Taube C,Wiedenroth C B Pneumologie (Stuttgart, Germany) Kongenitale Pulmonalarterienstenosen sind eine seltene Ursache der pulmonalen Hypertonie (PH). Die Erkrankung wird in ihrer Häufigkeit vermutlich unterschätzt, und sie sollte in der Abklärung einer PH bedacht werden.Die Vorstellung einer 43-jährigen Patientin erfolgte zur Therapieoptimierung und Evaluation einer möglichen Lungentransplantation mit der Arbeitsdiagnose kongenitale Pulmonalarterienstenosen.Die Patientin beklagte eine seit der frühen Kindheit bestehende Belastungsdyspnoe aktuell entsprechend WHO-FC-Klasse II-III.Die Krankengeschichte zeigte die Erstdiagnose einer primären pulmonalarteriellen Hypertonie (IPAH) vor 17 Jahren. Es erfolgte eine PH-spezifische Medikation in wechselnden Kombinationen. Im Rahmen eines Zentrumswechsels erfolgte eine Reevaluation, und bei Nachweis eines typischen Mismatch mit normaler Ventilation, jedoch keilförmig gestörter Perfusion in der Lungenszintigrafie wurde eine chronisch thromboembolische pulmonale Hypertonie (CTEPH) vermutet. Die Pulmonalis-Angiografie zeigte ausschließlich subsegmental gelegene Stenosierungen sowie Gefäßabbrüche mit korrespondierenden Minderperfusionen, passend zu einer CTEPH. Im Rahmen der ersten Intervention erfolgte aufgrund der ungewöhnlichen Morphologie der pulmonalarteriellen Läsionen eine Erweiterung der Diagnostik mittels optischer Kohärenztomografie (OCT). Bei der Patientin fand sich kein endoluminales Material, jedoch eine kräftige Gefäßwand. Damit wurde die Diagnose einer pumonalen Hypertonie bei kongenitalen Pulmonalarterienstenosen mit In-situ-Thrombosierung gestellt. 10.1055/a-1362-4996
    Lung tissue remodelling in MCT-induced pulmonary hypertension: a proposal for a novel scoring system and changes in extracellular matrix and fibrosis associated gene expression. Franz Marcus,Grün Katja,Betge Stefan,Rohm Ilonka,Ndongson-Dongmo Bernadin,Bauer Reinhard,Schulze P Christian,Lichtenauer Michael,Petersen Iver,Neri Dario,Berndt Alexander,Jung Christian Oncotarget Pulmonary hypertension (PH) is associated with vasoconstriction and remodelling. We studied lung tissue remodelling in a rat model of PH with special focus on histology and extracellular matrix (ECM) remodelling. After induction of PH by monocrotaline, lung tissue was analysed histologically, by gene expression analysis and immunofluorescence labelling of ED-A domain containing fibronectin (ED-A+ Fn), B domain containing tenascin-C (B+ Tn-C) as well as alpha-smooth muscle actin (α-SMA). Serum concentrations of ED-A+ Fn were determined by ELISA. Systolic right ventricular pressure (RVPsys) values were significantly elevated in PH (n = 18; 75 ± 26.4 mmHg) compared to controls (n = 10; 29 ± 19.3 mmHg; p = 0.015). The histological sum-score was significantly increased in PH (8.0 ± 2.2) compared to controls (2.5 ± 1.6; p < 0.001). Gene expression analysis revealed relevant induction of several key genes of extracellular matrix remodelling. Increased protein deposition of ED-A+ Fn but not of B+ Tn-C and α-SMA in lung tissue was found in PH (2.88 ± 3.19 area%) compared to controls (1.32 ± 0.16 area%; p = 0.030). Serum levels of ED-A+ Fn were significantly higher in PH (p = 0.007) positively correlating with RVPsys (r = 0.618, p = 0.019). We here present a novel histological scoring system to assess lung tissue remodelling in PH. Gene expression analysis revealed induction of candidate genes involved in collagen matrix turnover, fibrosis and vascular remodelling. The stable increased tissue deposition of ED-A+ Fn in PH as well as its dynamics in serum suggests a role as a promising novel biomarker and potential therapeutic target. 10.18632/oncotarget.13220
    Concurrent Molecular Magnetic Resonance Imaging of Inflammatory Activity and Extracellular Matrix Degradation for the Prediction of Aneurysm Rupture. Brangsch Julia,Reimann Carolin,Kaufmann Jan O,Adams Lisa C,Onthank David C,Thöne-Reineke Christa,Robinson Simon P,Buchholz Rebecca,Karst Uwe,Botnar Rene M,Hamm Bernd,Makowski Marcus R Circulation. Cardiovascular imaging BACKGROUND:Molecular magnetic resonance imaging is a promising modality for the characterization of abdominal aortic aneurysms (AAAs). The combination of different molecular imaging biomarkers may improve the assessment of the risk of rupture. This study investigates the feasibility of imaging inflammatory activity and extracellular matrix degradation by concurrent dual-probe molecular magnetic resonance imaging in an AAA mouse model. METHODS:Osmotic minipumps with a continuous infusion of Ang II (angiotensin II; 1000 ng/[kg·min]) to induce AAAs were implanted in apolipoprotein-deficient mice (N=58). Animals were assigned to 2 groups. In group 1 (longitudinal group, n=13), imaging was performed once after 1 week with a clinical dose of a macrophage-specific iron oxide-based probe (ferumoxytol, 4 mgFe/kg, surrogate marker for inflammatory activity) and an elastin-specific gadolinium-based probe (0.2 mmol/kg, surrogate marker for extracellular matrix degradation). Animals were then monitored with death as end point. In group 2 (week-by-week-group), imaging with both probes was performed after 1, 2, 3, and 4 weeks (n=9 per group). Both probes were evaluated in 1 magnetic resonance session. RESULTS:The combined assessment of inflammatory activity and extracellular matrix degradation was the strongest predictor of AAA rupture (sensitivity 100%; specificity 89%; area under the curve, 0.99). Information from each single probe alone resulted in lower predictive accuracy. In vivo measurements for the elastin- and iron oxide-probe were in good agreement with ex vivo histopathology (Prussian blue-stain: R=0.96, P<0.001; Elastica van Giesson stain: R=0.79, P<0.001). Contrast-to-noise ratio measurements for the iron oxide and elastin-probe were in good agreement with inductively coupled mass spectroscopy ( R=0.88, R=0.75, P<0.001) and laser ablation coupled to inductively coupled plasma-mass spectrometry. CONCLUSIONS:This study demonstrates the potential of the concurrent assessment of inflammatory activity and extracellular matrix degradation by dual-probe molecular magnetic resonance imaging in an AAA mouse model. Based on the combined information from both molecular probes, the rupture of AAAs could reliably be predicted. 10.1161/CIRCIMAGING.118.008707
    In utero estrogenic endocrine disruption alters the stroma to increase extracellular matrix density and mammary gland stiffness. Wormsbaecher Clarissa,Hindman Andrea R,Avendano Alex,Cortes-Medina Marcos,Jones Caitlin E,Bushman Andrew,Onua Lotanna,Kovalchin Claire E,Murphy Alina R,Helber Hannah L,Shapiro Ali,Voytovitch Kyle,Kuang Xingyan,Aguilar-Valenzuela Renan,Leight Jennifer L,Song Jonathan W,Burd Craig J Breast cancer research : BCR BACKGROUND:In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. METHODS:We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. RESULTS:We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. CONCLUSIONS:As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood. 10.1186/s13058-020-01275-w
    Glycoproteomic Analysis of the Aortic Extracellular Matrix in Marfan Patients. Yin 殷晓科 Xiaoke,Wanga Shaynah,Fellows Adam L,Barallobre-Barreiro Javier,Lu Ruifang,Davaapil Hongorzul,Franken Romy,Fava Marika,Baig Ferheen,Skroblin Philipp,Xing Qiuru,Koolbergen David R,Groenink Maarten,Zwinderman Aeilko H,Balm Ron,de Vries Carlie J M,Mulder Barbara J M,Viner Rosa,Jahangiri Marjan,Reinhardt Dieter P,Sinha Sanjay,de Waard Vivian,Mayr Manuel Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:Marfan syndrome (MFS) is caused by mutations in FBN1 (fibrillin-1), an extracellular matrix (ECM) component, which is modified post-translationally by glycosylation. This study aimed to characterize the glycoproteome of the aortic ECM from patients with MFS and relate it to aortopathy. Approach and Results: ECM extracts of aneurysmal ascending aortic tissue from patients with and without MFS were enriched for glycopeptides. Direct N-glycopeptide analysis by mass spectrometry identified 141 glycoforms from 47 glycosites within 35 glycoproteins in the human aortic ECM. Notably, MFAP4 (microfibril-associated glycoprotein 4) showed increased and more diverse N-glycosylation in patients with MFS compared with control patients. MFAP4 mRNA levels were markedly higher in MFS aortic tissue. MFAP4 protein levels were also increased at the predilection (convexity) site for ascending aorta aneurysm in bicuspid aortic valve patients, preceding aortic dilatation. In human aortic smooth muscle cells, MFAP4 mRNA expression was induced by TGF (transforming growth factor)-β1 whereas siRNA knockdown of MFAP4 decreased FBN1 but increased elastin expression. These ECM changes were accompanied by differential gene expression and protein abundance of proteases from ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family and their proteoglycan substrates, respectively. Finally, high plasma MFAP4 concentrations in patients with MFS were associated with a lower thoracic descending aorta distensibility and greater incidence of type B aortic dissection during 68 months follow-up. CONCLUSIONS:Our glycoproteomics analysis revealed that MFAP4 glycosylation is enhanced, as well as its expression during the advanced, aneurysmal stages of MFS compared with control aneurysms from patients without MFS. 10.1161/ATVBAHA.118.312175
    Chronic mild hypoxia promotes profound vascular remodeling in spinal cord blood vessels, preferentially in white matter, via an α5β1 integrin-mediated mechanism. Halder Sebok K,Kant Ravi,Milner Richard Angiogenesis Spinal cord injury (SCI) leads to rapid destruction of neuronal tissue, resulting in devastating motor and sensory deficits. This is exacerbated by damage to spinal cord blood vessels and loss of vascular integrity. Thus, approaches that protect existing blood vessels or stimulate the growth of new blood vessels might present a novel approach to minimize loss or promote regeneration of spinal cord tissue following SCI. In light of the remarkable power of chronic mild hypoxia (CMH) to stimulate vascular remodeling in the brain, the goal of this study was to examine how CMH (8% O for up to 7 days) affects blood vessel remodeling in the spinal cord. We found that CMH promoted the following: (1) endothelial proliferation and increased vascularity as a result of angiogenesis and arteriogenesis, (2) increased vascular expression of the angiogenic extracellular matrix protein fibronectin as well as concomitant increases in endothelial expression of the fibronectin receptor α5β1 integrin, (3) strongly upregulated endothelial expression of the tight junction proteins claudin-5, ZO-1 and occludin and (4) astrocyte activation. Of note, the vascular remodeling changes induced by CMH were more extensive in white matter. Interestingly, hypoxic-induced vascular remodeling in spinal cord blood vessels was markedly attenuated in mice lacking endothelial α5 integrin expression (α5-EC-KO mice). Taken together, these studies demonstrate the considerable remodeling potential of spinal cord blood vessels and highlight an important angiogenic role for the α5β1 integrin in promoting endothelial proliferation. They also imply that stimulation of the α5β1 integrin or controlled use of mild hypoxia might provide new approaches for promoting angiogenesis and improving vascular integrity in spinal cord blood vessels. 10.1007/s10456-017-9593-2
    BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation. Chang Ziyi,Zhao Guizhen,Zhao Yang,Lu Haocheng,Xiong Wenhao,Liang Wenying,Sun Jinjian,Wang Huilun,Zhu Tianqing,Rom Oren,Guo Yanhong,Fan Yanbo,Chang Lin,Yang Bo,Garcia-Barrio Minerva T,Lin Jiandie D,Chen Y Eugene,Zhang Jifeng Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA. CONCLUSIONS:Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA. 10.1161/ATVBAHA.120.314955
    Wild-type amyloid beta 1-40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity. Blaise Régis,Mateo Véronique,Rouxel Clotilde,Zaccarini François,Glorian Martine,Béréziat Gilbert,Golubkov Vladislav S,Limon Isabelle Aging cell Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-β (Aβ) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aβ-peptide deposits [composed mainly of wild-type (WT) Aβ(1-40) peptide in sporadic forms] induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated with their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and microhemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of matrix metalloprotease (MMP)-2 activation in WT-Aβ(1-40) -treated VSMC and provide evidence that MMP activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the WT-Aβ(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity nor hampering the expression of membrane type1-MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aβ(1-40) -induced cell apoptosis. Our results, in contrast to those obtained using the Aβ(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA. 10.1111/j.1474-9726.2012.00797.x
    Fibulin-4 is essential for maintaining arterial wall integrity in conduit but not muscular arteries. Halabi Carmen M,Broekelmann Thomas J,Lin Michelle,Lee Vivian S,Chu Mon-Li,Mecham Robert P Science advances Homozygous or compound heterozygous mutations in fibulin-4 () lead to autosomal recessive cutis laxa type 1B (ARCL1B), a multisystem disorder characterized by significant cardiovascular abnormalities, including abnormal elastin assembly, arterial tortuosity, and aortic aneurysms. We sought to determine the consequences of a human disease-causing mutation in (E57K) on the cardiovascular system and vascular elastic fibers in a mouse model of ARCL1B. mice were hypertensive and developed arterial elongation, tortuosity, and ascending aortic aneurysms. Smooth muscle cell organization within the arterial wall of large conducting vessels was abnormal, and elastic fibers were fragmented and had a moth-eaten appearance. In contrast, vessel wall structure and elastic fiber integrity were normal in resistance/muscular arteries (renal, mesenteric, and saphenous). Elastin cross-linking and total elastin content were unchanged in large or small arteries, whereas elastic fiber architecture was abnormal in large vessels. While the E57K mutation did not affect mRNA levels, FBLN4 protein was lower in the ascending aorta of mutant animals compared to wild-type arteries but equivalent in mesenteric arteries. We found a differential role of FBLN4 in elastic fiber assembly, where it functions mainly in large conduit arteries. These results suggest that elastin assembly has different requirements depending on vessel type. Normal levels of elastin cross-links in mutant tissue call into question FBLN4's suggested role in mediating lysyl oxidase-elastin interactions. Future studies investigating tissue-specific elastic fiber assembly may lead to novel therapeutic interventions for ARCL1B and other disorders of elastic fiber assembly. 10.1126/sciadv.1602532
    The molecular mechanism of mechanotransduction in vascular homeostasis and disease. Yamashiro Yoshito,Yanagisawa Hiromi Clinical science (London, England : 1979) Blood vessels are constantly exposed to mechanical stimuli such as shear stress due to flow and pulsatile stretch. The extracellular matrix maintains the structural integrity of the vessel wall and coordinates with a dynamic mechanical environment to provide cues to initiate intracellular signaling pathway(s), thereby changing cellular behaviors and functions. However, the precise role of matrix-cell interactions involved in mechanotransduction during vascular homeostasis and disease development remains to be fully determined. In this review, we introduce hemodynamics forces in blood vessels and the initial sensors of mechanical stimuli, including cell-cell junctional molecules, G-protein-coupled receptors (GPCRs), multiple ion channels, and a variety of small GTPases. We then highlight the molecular mechanotransduction events in the vessel wall triggered by laminar shear stress (LSS) and disturbed shear stress (DSS) on vascular endothelial cells (ECs), and cyclic stretch in ECs and vascular smooth muscle cells (SMCs)-both of which activate several key transcription factors. Finally, we provide a recent overview of matrix-cell interactions and mechanotransduction centered on fibronectin in ECs and thrombospondin-1 in SMCs. The results of this review suggest that abnormal mechanical cues or altered responses to mechanical stimuli in EC and SMCs serve as the molecular basis of vascular diseases such as atherosclerosis, hypertension and aortic aneurysms. Collecting evidence and advancing knowledge on the mechanotransduction in the vessel wall can lead to a new direction of therapeutic interventions for vascular diseases. 10.1042/CS20190488
    Role of extracellular matrix in the pathogenesis of pulmonary arterial hypertension. Thenappan Thenappan,Chan Stephen Y,Weir E Kenneth American journal of physiology. Heart and circulatory physiology Pulmonary arterial hypertension (PAH) is characterized by remodeling of the extracellular matrix (ECM) of the pulmonary arteries with increased collagen deposition, cross-linkage of collagen, and breakdown of elastic laminae. Extracellular matrix remodeling occurs due to an imbalance in the proteolytic enzymes, such as matrix metalloproteinases, elastases, and lysyl oxidases, and tissue inhibitor of matrix metalloproteinases, which, in turn, results from endothelial cell dysfunction, endothelial-to-mesenchymal transition, and inflammation. ECM remodeling and pulmonary vascular stiffness occur early in the disease process, before the onset of the increase in the intimal and medial thickness and pulmonary artery pressure, suggesting that the ECM is a cause rather than a consequence of distal pulmonary vascular remodeling. ECM remodeling and increased pulmonary arterial stiffness promote proliferation of pulmonary vascular cells (endothelial cells, smooth muscle cells, and adventitial fibroblasts) through mechanoactivation of various signaling pathways, including transcriptional cofactors YAP/TAZ, transforming growth factor-β, transient receptor potential channels, Toll-like receptor, and NF-κB. Inhibition of ECM remodeling and mechanotransduction prevents and reverses experimental pulmonary hypertension. These data support a central role for ECM remodeling in the pathogenesis of the PAH, making it an attractive novel therapeutic target. 10.1152/ajpheart.00136.2018
    Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins. Barallobre-Barreiro Javier,Oklu Rahmi,Lynch Marc,Fava Marika,Baig Ferheen,Yin Xiaoke,Barwari Temo,Potier David N,Albadawi Hassan,Jahangiri Marjan,Porter Karen E,Watkins Michael T,Misra Sanjay,Stoughton Julianne,Mayr Manuel Cardiovascular research AIMS:Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. METHODS AND RESULTS:To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. CONCLUSION:The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis. 10.1093/cvr/cvw075
    The NuRD chromatin-remodeling enzyme CHD4 promotes embryonic vascular integrity by transcriptionally regulating extracellular matrix proteolysis. Ingram Kyle G,Curtis Carol D,Silasi-Mansat Robert,Lupu Florea,Griffin Courtney T PLoS genetics The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4--a catalytic subunit of NuRD complexes--died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development. 10.1371/journal.pgen.1004031
    Knockdown of lncRNA Inhibits Vascular Smooth Muscle Cell Apoptosis and Extracellular Matrix Disruption in a Murine Abdominal Aortic Aneurysm Model. Zhang Zhidong,Zou Gangqiang,Chen Xiaosan,Lu Wei,Liu Jianyang,Zhai Shuiting,Qiao Gang Molecules and cells This study was designed to determine the effects of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 () on vascular smooth muscle cell (VSMC) apoptosis and extracellular matrix (ECM) disruption in a murine abdominal aortic aneurysm (AAA) model. After injection of -silencing lentiviruses, AAA was induced in Apolipoprotein E-deficient (ApoE) male mice by angiotensin II (Ang II) infusion for four weeks. After Ang II infusion, mouse serum levels of pro-inflammatory cytokines were analysed, and aortic tissues were isolated for histological, RNA, and protein analysis. Our results also showed that expression was significantly upregulated in abdominal aortic tissues from AAA patients compared with that in controls. Additionally, Ang II treatment significantly increased expression, both in cultured mouse VSMCs and in AAA murine abdominal aortic tissues. Of note, the effects of Ang II in facilitating cell apoptosis, increasing matrix metalloproteinase (MMP)-2 and MMP-9, reducing tissue inhibitor of MMP (TIMP)-1, and promoting switching from the contractile to synthetic phenotype in cultured VSMCs were enhanced by overexpression of but attenuated by knockdown of . Furthermore, knockdown of reversed Ang II-induced AAA-associated alterations in mice, as evidenced by attenuation of aortic diameter dilation, marked adventitial thickening, loss of elastin in the aorta, enhanced aortic cell apoptosis, elevated MMP-2 and MMP-9, reduced TIMP-1, and increased pro-inflammatory cytokines. In conclusion, our findings demonstrate that knockdown of lncRNA suppresses VSMC apoptosis, ECM disruption, and serum pro-inflammatory cytokines in a murine Ang II-induced AAA model. 10.14348/molcells.2018.0162
    The role of extracellular matrix in retinal vascular development and preretinal neovascularization. Bishop Paul N Experimental eye research Extracellular matrix (ECM) plays a central role in angiogenesis. ECM degrading enzymes breakdown the pre-existing vascular basement membrane at an early stage of angiogenesis and subsequently degrade stromal ECM as the new vessels invade into tissues. Conversely certain ECM components including collagen, fibronectin or fibrin are required for endothelial cell migration and tube morphogenesis. As the new vessels form they lay down a basement membrane that surrounds the endothelial tubes and is essential for their stability. In the rodent eye the transient expression of fibronectin and matricellular proteins plays a key role in retinal vascular development. In pathological retinal angiogenesis, such as in proliferative diabetic retinopathy, preretinal neovascularization occurs where new blood vessels invade the cortical vitreous gel and these blood vessels require vitreous collagen for their growth. The vitreous is normally anti-angiogenic and contains endogenous ECM inhibitors of angiogenesis including opticin and thombospondins, and ECM fragments such as endostatin. In preretinal neovascularization, the combined anti-angiogenic effects of these molecules are overcome by an excess of growth factors such as vascular endothelial growth factor-A, and new vessels grow into the vitreous with potentially blinding sequelae. 10.1016/j.exer.2014.10.021
    Endometrial vascular development in heavy menstrual bleeding: altered spatio-temporal expression of endothelial cell markers and extracellular matrix components. Biswas Shivhare Sourima,Bulmer Judith N,Innes Barbara A,Hapangama Dharani K,Lash Gendie E Human reproduction (Oxford, England) STUDY QUESTION:Are there any phenotypic and structural/architectural changes in the vessels of endometrium and superficial myometrium during the normal menstrual cycle in healthy women and those with heavy menstrual bleeding (HMB)? SUMMARY ANSWER:Spatial and temporal differences in protein levels of endothelial cell (EC) markers and components of the extracellular matrix (ECM) were detected across the menstrual cycle in healthy women and these are altered in HMB. WHAT IS KNOWN ALREADY:HMB affects 30% of women of reproductive age with ~50% of cases being idiopathic. We have previously shown that the differentiation status of endometrial vascular smooth muscle cells (VSMCs) is altered in women with HMB, suggesting altered vessel maturation compared to controls. Endometrial arteriogenesis requires the co-ordinated maturation not only of the VSMCs but also the underlying ECs and surrounding ECM. We hypothesized that there are spatial and temporal patterns of protein expression of EC markers and vascular ECM components in the endometrium across the menstrual cycle, which are altered in women with HMB. STUDY DESIGN, SIZE, DURATION:Biopsies containing endometrium and superficial myometrium were taken from hysterectomy specimens from both healthy control women without endometrial pathology and women with subjective HMB in the proliferative (PP), early secretory (ESP), mid secretory (MSP) and late secretory (LSP) phases (N = 5 for each cycle phase and subject group). Samples were fixed in formalin and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS:Serial sections (3μm thick) were immunostained for EC markers (factor VIII related antigen (F8RA), CD34, CD31 and ulex europaeus-agglutinin I (UEA-1) lectin), structural ECM markers (osteopontin, laminin, fibronectin and collagen IV) and for Ki67 to assess proliferation. Immunoreactivity of vessels in superficial myometrium, endometrial stratum basalis, stratum functionalis and luminal region was scored using either a modified Quickscore or by counting the number of positive vessels. MAIN RESULTS AND THE ROLE OF CHANCE:In control samples, all four EC markers showed a dynamic expression pattern according to the menstrual cycle phase, in both endometrial and myometrial vessels. EC protein marker expression was altered in women with HMB compared with controls, especially in the secretory phase in the endometrial luminal region and stratum functionalis. For example, in the LSP expression of UEA-1 and CD31 in the luminal region decreased in HMB (mean quickscore: 1 and 5, respectively) compared with controls (3.2 and 7.4, respectively) (both P = 0.008), while expression of F8RA and CD34 increased in HMB (1.4 and 8, respectively) compared with controls (0 and 5.8, respectively) (both P = 0.008). There was also a distinct pattern of expression of the vascular structural ECM protein components osteopontin, laminin, fibronectin and collagen IV in the superficial myometrium, stratum functionalis and stratum basalis during the menstrual cycle, which was altered in HMB. In particular, compared with controls, osteopontin expression in HMB was higher in stratum functionalis in the LSP (7.2 and 11.2, respectively P = 0.008), while collagen IV expression was reduced in stratum basalis in the MSP (4.6 and 2.8, respectively P = 0.002) and in stratum functionalis in the ESP (7 and 3.2, respectively P = 0.008). LIMITATIONS, REASONS FOR CAUTION:The protein expression of vascular EC markers and ECM components was assessed using a semi-quantitative approach in both straight and spiral arterioles. In our hospital, HMB is determined by subjective criteria and levels of blood loss were not assessed. WIDER IMPLICATIONS OF THE FINDINGS:Variation in the protein expression pattern between the four EC markers highlights the importance of choice of EC marker for investigation of endometrial vessels. Differences in expression of the different EC markers may reflect developmental stage dependent expression of EC markers in endometrial vessels, and their altered expression in HMB may reflect dysregulated vascular development. This hypothesis is supported by altered expression of ECM proteins within endometrial vessel walls, as well as our previous data showing a dysregulation in VSMC contractile protein expression in the endometrium of women with HMB. Taken together, these data support the suggestion that HMB symptoms are associated with weaker vascular structures, particularly in the LSP of the menstrual cycle, which may lead to increased and extended blood flow during menstruation. STUDY FUNDING/COMPETING INTEREST(S):This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. TRIAL REGISTRATION NUMBER:Not applicable. 10.1093/humrep/dex378
    Elastic fibres and vascular structure in hypertension. Arribas Silvia M,Hinek Aleksander,González M Carmen Pharmacology & therapeutics Blood vessels are dynamic structures composed of cells and extracellular matrix (ECM), which are in continuous cross-talk with each other. Thus, cellular changes in phenotype or in proliferation/death rate affect ECM synthesis. In turn, ECM elements not only provide the structural framework for vascular cells, but they also modulate cellular function through specific receptors. These ECM-cell interactions, together with neurotransmitters, hormones and the mechanical forces imposed by the heart, modulate the structural organization of the vascular wall. It is not surprising that pathological states related to alterations in the nervous, humoral or haemodynamic environment-such as hypertension-are associated with vascular wall remodeling, which, in the end, is deleterious for cardiovascular function. However, the question remains whether these structural alterations are simply a consequence of the disease or if there are early cellular or ECM alterations-determined either genetically or by environmental factors-that can predispose to vascular remodeling independent of hypertension. Elastic fibres might be key elements in the pathophysiology of hypertensive vascular remodeling. In addition to the well known effects of hypertension on elastic fibre fatigue and accelerated degradation, leading to loss of arterial wall resilience, recent investigations have highlighted new roles for individual components of elastic fibres and their degradation products. These elements can act as signal transducers and regulate cellular proliferation, migration, phenotype, and ECM degradation. In this paper, we review current knowledge regarding components of elastic fibres and discuss their possible pathomechanistic associations with vascular structural abnormalities and with hypertension development or progression. 10.1016/j.pharmthera.2005.12.003
    Inhibition of apoptosis exacerbates fatigue-damage tendon injuries in an in vivo rat model. Bell R,Robles-Harris M A,Anderson M,Laudier D,Schaffler M B,Flatow E L,Andarawis-Puri N European cells & materials Tendinopathy is a common and progressive musculoskeletal disease. Increased apoptosis is an end-stage tendinopathy manifestation, but its contribution to the pathology of the disease is unknown. A previously established in vivo model of fatigue damage accumulation shows that increased apoptosis is correlated with the severity of induced tendon damage, even in early onset of the disease, supporting its implication in the pathogenesis of the disease. Consequently, this study aimed to determine: (1) whether apoptosis could be inhibited after fatigue damage and (2) whether its inhibition could lead to remodeling of the extracellular matrix (ECM) and pericellular matrix (PCM), to ultimately improve the mechanical properties of fatigue-damaged tendons. The working hypothesis was that, despite the low vascular nature of the tendon, apoptosis would be inhibited, prompting increased production of matrix proteins and restoring tendon mechanical properties. Rats received 2 or 5 d of systemic pan-caspase inhibitor (Q-VD-OPh) or dimethyl sulfoxide (DMSO) carrier control injections starting immediately prior to fatigue loading and were sacrificed at days 7 and 14 post-fatigue-loading. Systemic pan-caspase inhibition for 2 d led to a surprising increase in apoptosis, but inhibition for 5 d increased the population of live cells that could repair the fatigue damage. Further analysis of the 5 d group showed that effective inhibition led to an increased population of cells producing ECM and PCM proteins, although typically in conjunction with oxidative stress markers. Ultimately, inhibition of apoptosis led to further deterioration in mechanical properties of fatigue-damaged tendons. 10.22203/eCM.v036a04
    Sox9 is increased in arterial plaque and stenosis, associated with synthetic phenotype of vascular smooth muscle cells and causes alterations in extracellular matrix and calcification. Augstein Antje,Mierke Johannes,Poitz David M,Strasser Ruth H Biochimica et biophysica acta. Molecular basis of disease Vascular smooth muscle cells (VSMC) exhibit a dual role in progression and maintenance of arteriosclerosis. They are fundamental for plaque stability but also can drive plaque progression. During pathogenic vascular remodeling, VSMC transdifferentiate into a phenotype with enhanced proliferation and migration. Moreover, they exert an increased capacity to generate extracellular matrix proteins. A special lineage of transdifferentiated VSMC expresses Sox9, a multi-functional transcription factor. The aim of the study was to examine the role of Sox9 in phenotypic alterations leading to arteriosclerosis. Using mouse models for arterial stenosis, Sox9 induction in diseased vessels was verified. The phenotypic switch of VSMC from contractile to proliferative nature caused a significant increase of Sox9 expression. Various factors known to be involved in the progression of arteriosclerosis were examined for their ability to modulate Sox9 expression in VSMC. While PDGF-BB resulted in a strong transient upregulation of Sox9, TGF-β1 appeared to be responsible for a moderate, but prolonged increase of Sox9 expression. Beside the regulation, functional studies focused on knockout and overexpression of Sox9. A Sox9-dependent alteration of extracellular matrix could be revealed and was associated with an upregulated calcium deposition. Taken together, Sox9 is identified as important factor of VSMC function by modulation the extracellular matrix composition and calcium deposition, which are important processes in plaque development. 10.1016/j.bbadis.2018.05.009
    Perivascular extracellular matrix hydrogels mimic native matrix microarchitecture and promote angiogenesis via basic fibroblast growth factor. Fercana George R,Yerneni Saigopalakrishna,Billaud Marie,Hill Jennifer C,VanRyzin Paul,Richards Tara D,Sicari Brian M,Johnson Scott A,Badylak Stephen F,Campbell Phil G,Gleason Thomas G,Phillippi Julie A Biomaterials Extracellular matrix (ECM)-derived bioscaffolds have been shown to elicit tissue repair through retention of bioactive signals. Given that the adventitia of large blood vessels is a richly vascularized microenvironment, we hypothesized that perivascular ECM contains bioactive signals that influence cells of blood vessel lineages. ECM bioscaffolds were derived from decellularized human and porcine aortic adventitia (hAdv and pAdv, respectively) and then shown have minimal DNA content and retain elastin and collagen proteins. Hydrogel formulations of hAdv and pAdv ECM bioscaffolds exhibited gelation kinetics similar to ECM hydrogels derived from porcine small intestinal submucosa (pSIS). hAdv and pAdv ECM hydrogels displayed thinner, less undulated, and fibrous microarchitecture reminiscent of native adventitia, with slight differences in ultrastructure visible in comparison to pSIS ECM hydrogels. Pepsin-digested pAdv and pSIS ECM bioscaffolds increased proliferation of human adventitia-derived endothelial cells and this effect was mediated in part by basic fibroblast growth factor (FGF2). Human endothelial cells cultured on Matrigel substrates formed more numerous and longer tube-like structures when supplemented with pAdv ECM bioscaffolds, and FGF2 mediated this matrix signaling. ECM bioscaffolds derived from pAdv promoted FGF2-dependent in vivo angiogenesis in the chick chorioallantoic membrane model. Using an angiogenesis-focused protein array, we detected 55 angiogenesis-related proteins, including FGF2 in hAdv, pAdv and pSIS ECMs. Interestingly, 19 of these factors were less abundant in ECMs bioscaffolds derived from aneurysmal specimens of human aorta when compared with non-aneurysmal (normal) specimens. This study reveals that Adv ECM hydrogels recapitulate matrix fiber microarchitecture of native adventitia, and retain angiogenesis-related actors and bioactive properties such as FGF2 signaling capable of influencing processes important for angiogenesis. This work supports the use of Adv ECM bioscaffolds for both discovery biology and potential translation towards microvascular regeneration in clinical applications. 10.1016/j.biomaterials.2017.01.037
    Extracellular matrix inflammation in vascular cognitive impairment and dementia. Rosenberg Gary A Clinical science (London, England : 1979) Vascular cognitive impairment and dementia (VCID) include a wide spectrum of chronic manifestations of vascular disease related to large vessel strokes and small vessel disease (SVD). Lacunar strokes and white matter (WM) injury are consequences of SVD. The main vascular risk factor for SVD is brain hypoperfusion from cerebral blood vessel narrowing due to chronic hypertension. The hypoperfusion leads to activation and degeneration of astrocytes with the resulting fibrosis of the extracellular matrix (ECM). Elasticity is lost in fibrotic cerebral vessels, reducing the response of stiffened blood vessels in times of increased metabolic need. Intermittent hypoxia/ischaemia activates a molecular injury cascade, producing an incomplete infarction that is most damaging to the deep WM, which is a watershed region for cerebral blood flow. Neuroinflammation caused by hypoxia activates microglia/macrophages to release proteases and free radicals that perpetuate the damage over time to molecules in the ECM and the neurovascular unit (NVU). Matrix metalloproteinases (MMPs) secreted in an attempt to remodel the blood vessel wall have the undesired consequences of opening the blood-brain barrier (BBB) and attacking myelinated fibres. This dual effect of the MMPs causes vasogenic oedema in WM and vascular demyelination, which are the hallmarks of the subcortical ischaemic vascular disease (SIVD), which is the SVD form of VCID also called Binswanger's disease (BD). Unravelling the complex pathophysiology of the WM injury-related inflammation in the small vessel form of VCID could lead to novel therapeutic strategies to reduce damage to the ECM, preventing the progressive damage to the WM. 10.1042/CS20160604
    Extracellular matrix scaffolding in angiogenesis and capillary homeostasis. Marchand Marion,Monnot Catherine,Muller Laurent,Germain Stéphane Seminars in cell & developmental biology The extracellular matrix (ECM) of blood vessels, which is composed of both the vascular basement membrane (BM) and the interstitial ECM is identified as a crucial component of the vasculature. We here focus on the unique molecular composition and scaffolding of the capillary ECM, which provides structural support to blood vessels and regulates properties of endothelial cells and pericytes. The major components of the BM are collagen IV, laminins, heparan sulfate proteoglycans and nidogen and also associated proteins such as collagen XVIII and fibronectin. Their organization and scaffolding in the BM is required for proper capillary morphogenesis and maintenance of vascular homeostasis. The BM also regulates vascular mechanosensing. A better understanding of the mechanical and structural properties of the vascular BM and interstitial ECM therefore opens new perspectives to control physiological and pathological angiogenesis and vascular homeostasis. The overall aim of this review is to explain how ECM scaffolding influences angiogenesis and capillary integrity. 10.1016/j.semcdb.2018.08.007
    A hydrogel derived from acellular blood vessel extracellular matrix to promote angiogenesis. Fu Wei,Xu Peng,Feng Bei,Lu Yang,Bai Jie,Zhang Jialiang,Zhang Wenjie,Yin Meng Journal of biomaterials applications The biocompatibility and bioactivity of injectable acellular extracellular matrix nominates its use as an optimal candidate for cell delivery, serving as a reconstructive scaffold. In this study, we investigated the feasibility of preparing a blood vessel matrix (BVM) hydrogel, which revealed its pro-angiogenic effects in vitro and its therapeutic effects in an in vivo skin flap model. Aortic and abdominal aortic arteries from pigs were acellularized by Triton-X 100 and confirmed by hematoxylin and eosin and 4,6-diamidino-2-phenylindole staining. Different concentrations of blood vessel matrix hydrogel were generated successfully through enzymatic digestion, neutralization, and gelation. Hematoxylin and eosin staining, Masson's trichrome staining, collagen type I immunohistochemistry staining, and enzyme-linked immunosorbent assays showed that type I collagen and some growth factors were retained in the hydrogel. Scanning electron microscopy demonstrated the different diametric fibrils in blood vessel matrix hydrogels. A blood vessel matrix hydrogel-coated plate promoted the tube formation of human umbilical vein endothelial cells in vitro. After injection into skin flaps, the hydrogel improved the flap survival rate and increased blood perfusion and capillary density. These results indicated that we successfully prepared a blood vessel matrix hydrogel and demonstrated its general characteristics and angiogenic effects in vitro and in vivo. 10.1177/0885328219831055
    Smooth muscle cell and arterial aging: basic and clinical aspects. Lacolley Patrick,Regnault Veronique,Avolio Alberto P Cardiovascular research Arterial aging engages a plethora of key signalling pathways that act in concert to induce vascular smooth muscle cell (VSMC) phenotypic changes leading to vascular degeneration and extracellular matrix degradation responsible for alterations of the mechanical properties of the vascular wall. This review highlights proof-of-concept examples of components of the extracellular matrix, VSMC receptors which connect extracellular and intracellular structures, and signalling pathways regulating changes in mechanotransduction and vascular homeostasis in aging. Furthermore, it provides a new framework for understanding how VSMC stiffness and adhesion to extracellular matrix contribute to arterial stiffness and how interactions with endothelial cells, platelets, and immune cells can regulate vascular aging. The identification of the key players of VSMC changes operating in large and small-sized arteries in response to increased mechanical load may be useful to better elucidate the causes and consequences of vascular aging and associated progression of hypertension, arteriosclerosis, and atherosclerosis. 10.1093/cvr/cvy009
    Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration. Sanyour Hanna J,Li Na,Rickel Alex P,Torres Haydee M,Anderson Ruthellen H,Miles Miranda R,Childs Josh D,Francis Kevin R,Tao Jianning,Hong Zhongkui The Journal of physiology KEY POINTS:This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. ABSTRACT:Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function. 10.1113/JP279528
    FOXO3a (Forkhead Transcription Factor O Subfamily Member 3a) Links Vascular Smooth Muscle Cell Apoptosis, Matrix Breakdown, Atherosclerosis, and Vascular Remodeling Through a Novel Pathway Involving MMP13 (Matrix Metalloproteinase 13). Yu Haixiang,Fellows Adam,Foote Kirsty,Yang Zhaoqing,Figg Nichola,Littlewood Trevor,Bennett Martin Arteriosclerosis, thrombosis, and vascular biology OBJECTIVE:Vascular smooth muscle cell (VSMC) apoptosis accelerates atherosclerosis and promotes breakdown of the extracellular matrix, but the mechanistic links between these 2 processes are unknown. The forkhead protein FOXO3a (forkhead transcription factor O subfamily member 3a) is activated in human atherosclerosis and induces a range of proapoptotic and other transcriptional targets. We, therefore, determined the mechanisms and consequences of FOXO3a activation in atherosclerosis and arterial remodeling after injury. APPROACH AND RESULTS:Expression of a conditional FOXO3a allele (FOXO3aA3ER) potently induced VSMC apoptosis, expression and activation of MMP13 (matrix metalloproteinase 13), and downregulation of endogenous TIMPs (tissue inhibitors of MMPs). and were direct FOXO3a transcriptional targets in VSMCs. Activation of endogenous FOXO3a also induced MMP13, extracellular matrix degradation, and apoptosis, and MMP13-specific inhibitors and fibronectin reduced FOXO3a-mediated apoptosis. FOXO3a activation in mice with VSMC-restricted FOXO3aA3ER induced MMP13 expression and activity and medial VSMC apoptosis. FOXO3a activation in FOXO3aA3ER/ApoE (apolipoprotein E deficient) mice increased atherosclerosis, increased necrotic core and reduced fibrous cap areas, and induced features of medial degeneration. After carotid artery ligation, FOXO3a activation increased VSMC apoptosis, VSMC proliferation, and neointima formation, all of which were reduced by MMP13 inhibition. CONCLUSIONS:FOXO3a activation induces VSMC apoptosis and extracellular matrix breakdown, in part, because of transcriptional activation of MMP13. FOXO3a activation promotes atherosclerosis and medial degeneration and increases neointima after injury that is partly dependent on MMP13. FOXO3a-induced MMP activation represents a direct mechanistic link between VSMC apoptosis and matrix breakdown in vascular disease. 10.1161/ATVBAHA.117.310502
    Upregulation of the actin cytoskeleton via myocardin leads to increased expression of type 1 collagen. Shi Zengdun,Rockey Don C Laboratory investigation; a journal of technical methods and pathology Liver fibrosis, a model wound healing system, is characterized by excessive deposition of extracellular matrix (ECM) in the liver. Although many fibrogenic cell types may express ECM, the hepatic stellate cell (HSC) is currently considered to be the major effector. HSCs transform into myofibroblast-like cells, also known as hepatic myofibroblasts in a process known as activation; this process is characterized in particular by de novo expression of smooth muscle alpha actin (SM α-actin) and type 1 collagen. The family of actins, which form the cell's cytoskeleton, are essential in many cellular processes. β-actin and cytoplasmic γ-actin (γ-actin) are ubiquitously expressed, whereas SM α-actin defines smooth muscle cell and myofibroblast phenotypes. Thus, SM α-actin is tightly associated with multiple functional properties. However, the regulatory mechanisms by which actin isoforms might regulate type 1 collagen remain unclear. In primary HSCs from normal and fibrotic rat liver, we demonstrate that myocardin, a canonical SRF cofactor, is upregulated in hepatic myofibroblasts and differentially regulates SM α-actin, γ-actin, and β-actins through activation of an ATTA box in the SM α-actin and a CCAAT box in γ-actin and β-actin promoters, respectively; moreover, myocardin differentially activated serum response factor (SRF) in CArG boxes of actin promoters. In addition, myocardin-stimulated Smad2 phosphorylation and RhoA expression, leading to increased expression of type 1 collagen in an actin cytoskeleton-dependent manner. Myocardin also directly enhanced SRF expression and stimulated collagen 1α1 and 1α2 promoter activities. In addition, overexpression of myocardin in vivo during carbon tetrachloride-induced liver injury led to increased HSC activation and fibrogenesis. In summary, our data suggest that myocardin plays a critical role in actin cytoskeletal dynamics during HSC activation, in turn, specifically regulating type I collagen expression in hepatic myofibroblasts. 10.1038/labinvest.2017.96
    Smooth muscle-specific TMEM16A expression protects against angiotensin II-induced cerebrovascular remodeling via suppressing extracellular matrix deposition. Zeng Xue-Lin,Sun Lu,Zheng Hua-Qing,Wang Guan-Lei,Du Yan-Hua,Lv Xiao-Fei,Ma Ming-Ming,Guan Yong-Yuan Journal of molecular and cellular cardiology Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca-activated Cl channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-β1/Smad3, and non-canonical TGF-β1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl-sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-β1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling. 10.1016/j.yjmcc.2019.07.002
    Temporary fatigue and altered extracellular matrix in skeletal muscle during progression of heart failure in rats. Rehn Tommy A,Borge Bengt A,Lunde Per K,Munkvik Morten,Sneve Marianne Lunde,Grøndahl Frøy,Aronsen Jan M,Sjaastad Ivar,Prydz Kristian,Kolset Svein O,Wiig Helge,Sejersted Ole M,Iversen Per O American journal of physiology. Regulatory, integrative and comparative physiology Patients with congestive heart failure (CHF) experience increased skeletal muscle fatigue. The mechanism underlying this phenomenon is unknown, but a deranged extracellular matrix (ECM) might be a contributing factor. Hence, we examined ECM components and regulators in a rat postinfarction model of CHF. At various time points during a 3.5 mo-period after induction of CHF in rats by left coronary artery ligation, blood, interstitial fluid (IF), and muscles were sampled. Isoflurane anesthesia was employed during all surgical procedures. IF was extracted by wicks inserted intermuscularly in a hind limb. We measured cytokines in plasma and IF, whereas matrix metalloproteinase (MMP) activity and collagen content, as well as the level of glycosaminoglycans and hyaluronan were determined in hind limb muscle. In vivo fatigue protocols of the soleus muscle were performed at 42 and 112 days after induction of heart failure. We found that the MMP activity and collagen content in the skeletal muscles increased significantly at 42 days after induction of CHF, and these changes were time related to increased skeletal muscle fatigability. These parameters returned to sham levels at 112 days. VEGF in IF was significantly lower in CHF compared with sham-operated rats at 3 and 10 days, but no difference was observed at 112 days. We conclude that temporary alterations in the ECM, possibly triggered by VEGF, are related to a transient development of skeletal muscle fatigue in CHF. 10.1152/ajpregu.90617.2008
    Tendon extracellular matrix damage, degradation and inflammation in response to in vitro overload exercise. Spiesz Ewa M,Thorpe Chavaunne T,Chaudhry Saira,Riley Graham P,Birch Helen L,Clegg Peter D,Screen Hazel R C Journal of orthopaedic research : official publication of the Orthopaedic Research Society The role of inflammation in tendon injury is uncertain and a topic of current interest. In vitro studies of tendon accelerated overload damage can serve as a valuable source of information on the early stages of tendinopathy. Viable fascicle bundles from bovine flexor tendons were subjected to cyclic uniaxial loading from 1-10% strain. Immuno-staining for inflammatory markers and matrix degradation markers was performed on the samples after mechanical testing. Loaded samples exhibited visible extracellular matrix damage, with disrupted collagen fibers and fiber kinks, and notable damage to the interfascicular matrix. Inflammatory markers COX-2 and IL-6 were only expressed in the cyclically loaded samples. Collagen degradation markers MMP-1 and C1,2C were colocalized in many areas, with staining occurring in the interfascicular matrix or the fascicular tenocytes. These markers were present in control samples, but staining became increasingly intense with loading. Little MMP-3 or MMP-13 was evident in control sections. In loaded samples, some sections showed intense staining of these markers, again localized to interfascicular regions. This study suggests that inflammatory markers may be expressed rapidly after tendon overload exercise. Interestingly, both inflammation and damage-induced matrix remodeling seem to be concentrated in, or in the vicinity of, the highly cellular interfascicular matrix. 10.1002/jor.22879