Intracellular Calreticulin Regulates Multiple Steps in Fibrillar Collagen Expression, Trafficking, and Processing into the Extracellular Matrix

AbstractFibrosis can affect any organ resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by expansion of connective tissue due to excessive deposition of extracellular matrix proteins (ECM), including the fibrillar forms of collagen. A significant hurdle for discovering cures for fibrosis is the lack of suitable models and techniques to quantify mature collagen deposition in tissues. Here we have extensively characterized an ex-vivo cultured human lung derived, precision-cut lung slices model (hPCLS) using live fluorescence light microscopy as well as mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint. Using an integrated approach of multiple readouts such as quantitative label-free Second Harmonic Generation (SHG) imaging to measure fibrillar collagen in the extracellular matrix and ELISA-based methods to measure soluble ECM biomarkers, we investigated TGFbeta1-mediated pro-fibrotic signalling in hPCLS. We demonstrate that hPCLS are viable and metabolically active with mesenchymal, epithelial, endothelial, and immune cells surviving for at least two weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed strong induction of ECM synthesis proteins P1NP and fibronectin upon TGFb stimulation. Importantly, this effect translated into an increased deposition of fibrillar collagen in ECM of cultured hPCLS as measured by a novel quantitative SHG-based imaging method only following addition of a metalloproteinase inhibitor (GM6001). Together the data show that an integrated approach of measuring soluble pro-fibrotic markers and quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.

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Abstract 124: Integrated Omics Approaches to Elucidate Targets of Cyclic AMP-Epac-Rap1A Signaling in Microvascular Smooth Muscle Cells: Identification of Role in Production and Deposition of Extracellular Matrix Fibrillar Collagen

Circulation Research ◽

10.1161/res.119.suppl_1.124 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Maqsood A Chotani

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle ◽

Cyclic Amp ◽

Blood Vessels ◽

Intracellular Signaling ◽

Small Gtpase ◽

Cytoskeletal Proteins ◽

Global Changes ◽

Fibrillar Collagen ◽

Healthy Human

Objective: Intracellular signaling by cyclic AMP (cAMP) has an essential role in vascular smooth muscle (VSM) physiology, including relaxation of large blood vessels such as aorta and pulmonary artery. Studies support a mechanism of signaling through e xchange p rotein a ctivated by c AMP (epac) and Ras-related small GTPase Rap1B down-regulation of RhoA activity. The role of cAMP in small blood vessels however was not examined, and remained unknown. This study elucidated the targets of cAMP signaling in peripheral blood vessels, specifically VSM explanted from healthy human (h) cutaneous arterioles and mouse (m) tail artery (microVSM). Results: Global changes in protein expression were assessed by Differential-in-Gel Electrophoresis in quiescent (h)-microVSM treated with the adenylate cyclase activator and cAMP elevating agent forskolin (10 μM, 30 min or 18 hr). Detectable changes were observed in proteins associated with the cytoskeleton, stress-response, protein-synthesis, -folding, -membrane transport, and extracellular matrix. Notably, actin modulating proteins caldesmon, ezrin-moesin, zyxin, gelsolin, and α-adducin, as well as cytoskeletal proteins, tubulin and vimentin were differentially expressed ( P< 0.05; 3-5 replicates). These results agreed with our recent demonstration of cAMP activation of epac-Rap1A, and RhoA-ROCK-F-actin signaling in (h)- and (m)-microVSM to increase expression and cell surface transport of functional α 2C -adrenoceptors (α 2C -ARs) that mediate vasoconstriction. Transcriptome analysis of Rap1A-null (knockout) (m)-microVSM transduced with constitutively active Rap1A showed connections to signaling linked to vessel production and deposition of extracellular matrix fibrillar collagen, compared with control cells ( P< 0.05, 4 replicates). Conclusions: Rap1 subtypes have a discrete role in the vasculature. This study links cAMP-Rap1A signaling to collagen and α 2C -AR expression in arteriole VSM. It suggests over-activation of Rap1A-coupled signaling in the peripheral circulation during chronic inflammation could lead to increased α 2C -AR mediated vessel reactivity, progressive perivascular fibrosis, and dysfunction as seen in human pathologies such as Raynaud’s phenomenon and scleroderma.

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Pectoralis Major (Breast) Muscle Extracellular Matrix Fibrillar Collagen Modifications Associated With the Wooden Breast Fibrotic Myopathy in Broilers

Frontiers in Physiology ◽

10.3389/fphys.2020.00461 ◽

2020 ◽

Vol 11 ◽

Cited By ~ 2

Author(s):

Sandra G. Velleman

Keyword(s):

Extracellular Matrix ◽

Pectoralis Major ◽

Breast Muscle ◽

Fibrillar Collagen ◽

Major Breast ◽

Wooden Breast

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Dense fibrillar collagen matrices to analyse extracellular matrix receptor function

Pathologie Biologie ◽

10.1016/j.patbio.2011.10.007 ◽

2012 ◽

Vol 60 (1) ◽

pp. 7-14 ◽

Cited By ~ 5

Author(s):

C. Helary ◽

B. Rodrigues-Sanchez ◽

S. Vigier ◽

M.-M. Giraud Guille

Keyword(s):

Extracellular Matrix ◽

Fibrillar Collagen ◽

Receptor Function ◽

Collagen Matrices

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Perlecan Maintains the Integrity of Cartilage and Some Basement Membranes

The Journal of Cell Biology ◽

10.1083/jcb.147.5.1109 ◽

1999 ◽

Vol 147 (5) ◽

pp. 1109-1122 ◽

Cited By ~ 413

Author(s):

Mercedes Costell ◽

Erika Gustafsson ◽

Attila Aszódi ◽

Matthias Mörgelin ◽

Wilhelm Bloch ◽

...

Keyword(s):

Extracellular Matrix ◽

Heart Function ◽

Basement Membranes ◽

Matrix Proteins ◽

Fibrillar Collagen ◽

Collagen Network ◽

Pericardial Cavity ◽

Cartilage Extracellular Matrix ◽

Elevated Expression ◽

The Brain

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

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Structural constraints on the evolution of the collagen fibril: convergence on a 1014-residue COL domain

Open Biology ◽

10.1098/rsob.140220 ◽

2015 ◽

Vol 5 (5) ◽

pp. 140220 ◽

Cited By ~ 8

Author(s):

David Anthony Slatter ◽

Richard William Farndale

Keyword(s):

Amino Acids ◽

Extracellular Matrix ◽

Triple Helix ◽

Type I Collagen ◽

Structural Features ◽

Structural Proteins ◽

Type I ◽

Structural Constraints ◽

Fibrillar Collagen ◽

Cross Linkage

Type I collagen is the fundamental component of the extracellular matrix. Its α1 gene is the direct descendant of ancestral fibrillar collagen and contains 57 exons encoding the rod-like triple-helical COL domain. We trace the evolution of the COL domain from a primordial collagen 18 residues in length to its present 1014 residues, the limit of its possible length. In order to maintain and improve the essential structural features of collagen during evolution, exons can be added or extended only in permitted, non-random increments that preserve the position of spatially sensitive cross-linkage sites. Such sites cannot be maintained unless the twist of the triple helix is close to 30 amino acids per turn. Inspection of the gene structure of other long structural proteins, fibronectin and titin, suggests that their evolution might have been subject to similar constraints.

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Thiol-ene cross-linked alginate hydrogel encapsulation modulates the extracellular matrix of kidney organoids by reducing abnormal type 1a1 collagen deposition

10.1101/2020.11.17.386250 ◽

2020 ◽

Author(s):

Thomas Geuens ◽

Floor A.A. Ruiter ◽

Anika Schumacher ◽

Francis L. C. Morgan ◽

Timo Rademakers ◽

...

Keyword(s):

Extracellular Matrix ◽

Renal Fibrosis ◽

Kidney Diseases ◽

Matrix Composition ◽

List Type ◽

Alginate Hydrogel ◽

Organoid Culture ◽

Collagen Expression ◽

Kidney Organoids

ABSTRACTDifferentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular, extracellular compositions and developmental plateau needs to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on a transwell filter. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.HIGHLIGHTSProlonging kidney organoid culture results in a developmental plateau instead of improved in vitro maturation.Proteomic analyses point to an increased expression of specific collagen subtypes associated with renal fibrosis.Encapsulating kidney organoids using a soft thiol-ene cross-linked alginate hydrogel reduces collagen type 1a1 and αSMA deposition.

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Probiotic Activity of Staphylococcus epidermidis Induces Collagen Type I Production through FFaR2/p-ERK Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms22031414 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1414

Author(s):

Indira Putri Negari ◽

Sunita Keshari ◽

Chun-Ming Huang

Keyword(s):

Extracellular Matrix ◽

Fatty Acid ◽

Carbon Source ◽

Staphylococcus Epidermidis ◽

Collagen Type ◽

Collagen Type I ◽

Type I ◽

Extrinsic Factors ◽

Erk Activation ◽

Collagen Expression

Collagen type I is a key structural component of dermis tissue and is produced by fibroblasts and the extracellular matrix. The skin aging process, which is caused by intrinsic or extrinsic factors, such as natural aging or free radical exposure, greatly reduces collagen expression, thereby leading to obstructed skin elasticity. We investigated the effective fermentation of Cetearyl isononanoate (CIN), a polyethylene glycol (PEG) analog, as a carbon source with the skin probiotic bacterium Staphylococcus epidermidis (S.epidermidis) or butyrate, as their fermentation metabolites could noticeably restore collagen expression through phosphorylated extracellular signal regulated kinase (p-ERK) activation in mouse fibroblast cells and skin. Both the in vitro and in vivo knockdown of short-chain fatty acid (SCFA) or free fatty acid receptor 2 (FFaR2) considerably blocked the probiotic effect of S. epidermidis on p-ERK-induced collagen type I induction. These results demonstrate that butyric acid (BA) in the metabolites of fermenting skin probiotic bacteria mediates FFaR2 to induce the synthesis of collagen through p-ERK activation. We hereby imply that metabolites from the probiotic S. epidermidis fermentation of CIN as a potential carbon source could restore impaired collagen in the dermal extracellular matrix (ECM), providing integrity and elasticity to skin.

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Preventive training does not interfere with mRNA-encoding myosin and collagen expression during pulmonary arterial hypertension

PLoS ONE ◽

10.1371/journal.pone.0244768 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0244768

Author(s):

Thaoan Bruno Mariano ◽

Anthony César de Souza Castilho ◽

Ana Karenina Dias de Almeida Sabela ◽

André Casanova de Oliveira ◽

Sarah Santiloni Cury ◽

...

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Functional Capacity ◽

Adaptation Period ◽

Cross Sectional ◽

Collagen Expression ◽

Pulmonary Arterial ◽

The Impact

To gain insight on the impact of preventive exercise during pulmonary arterial hypertension (PAH), we evaluated the gene expression of myosins and gene-encoding proteins associated with the extracellular matrix remodeling of right hypertrophied ventricles. We used 32 male Wistar rats, separated in four groups: Sedentary Control (S, n = 8); Control with Training (T, n = 8); Sedentary with Pulmonary Arterial Hypertension (SPAH, n = 8); and Pulmonary Arterial Hypertension with Training (TPAH, n = 8). All rats underwent a two-week adaptation period; T and TPAH group rats then proceeded to an eight-week training period on a treadmill. At the beginning of the 11th week, S and T groups received an intraperitoneal injection of saline, and SPAH and TPAH groups received an injection of monocrotaline (60 mg/kg). Rats in the T and TPAH groups then continued with the training protocol until the 13th week. We assessed exercise capacity, echocardiography analysis, Fulton’s index, cross-sectional areas of cardiomyocytes, collagen content and types, and fractal dimension (FD). Transcript abundance of myosins and extracellular matrix genes were estimated through reverse transcription-quantitative PCR (RT-qPCR). When compared to the SPAH group, the TPAH group showed increases in functional capacity and pulmonary artery acceleration time/pulmonary ejection time ratio and decreases in Fulton’s index and cross-sectional areas of myocyte cells. However, preventive exercise did not induce alterations in col1a1 and myh7 gene expression. Our findings demonstrate that preventive exercise improved functional capacity, reduced cardiac hypertrophy, and attenuated PH development without interfering in mRNA-encoding myosin and collagen expression during PAH.

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Differences in MMP-2 Expression in High- and Low-Grade MPNST and its Correlation with Prognostic Factors

Mutiara Medika Jurnal Kedokteran dan Kesehatan ◽

10.18196/mmjkk.v21i2.10882 ◽

2021 ◽

Vol 21 (2) ◽

pp. 71-78

Author(s):

Niniek Hardini ◽

Nurjati Chairani Siregar ◽

Puspita Eka Wuyung

Keyword(s):

Extracellular Matrix ◽

Spindle Cell ◽

Basal Membrane ◽

Low Grade ◽

Nerve Sheath Tumor ◽

High Grade ◽

Fibrillar Collagen ◽

Malignancy Grading ◽

Invasion Pathway ◽

The Relationship

Malignant peripheral nerve sheath tumor (MPNST) is a soft tissue sarcoma, which is difficult to distinguish from other spindle cell sarcomas. MPNST is hostile, with a high recurrence, and tends to metastasize hematogenously, especially to the lungs. A phase of the metastasis is a degradation of the extracellular matrix, where Matrix Metalloproteinase (MMP) plays an essential role in this process. Gelatinase-type MMP, MMP-2 and MMP-9, can degrade basal membrane and fibrillar collagen to open the invasion pathway. MMP-2 can degrade more collagen and non-collagen extracellular matrix than MMP-9. Therefore, the study aimed to see the relationship between MMP-2 overexpression and histopathological malignancy grading and other clinical prognostic variables. The study was conducted by immunohistochemical staining of MMP-2 in 39 cases, consisting of 19 cases of low-grade MPNST and 20 cases of high-grade MPNST. Subsequently, an analysis of the relationship between MMP-2 overexpression and the malignancy grading and clinical variables was performed, such as age, sex, and tumor size and location. MMP-2 overexpression was seen in 19 (95%) cases of high grade and three (15.8%) cases of low-grade MPNST (p 0.000). The study also found a significant relationship between MMP-2 overexpression and histopathology grading, which may be helpful to define the prognosis.

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